Methods and compositions for seamless cloning of nucleic acid molecules

ABSTRACT

The present invention is in the fields of biotechnology and molecular biology. More particularly, the present invention relates to cloning or subcloning one or more nucleic acid molecules comprising one or more type IIs restriction enzyme recognition sites. The present invention also embodies cloning such nucleic acid molecules using recombinational cloning methods such as those employing recombination sites and recombination proteins. The present invention also relates to nucleic acid molecules (including RNA and iRNA), as well as proteins, expressed from host cells produced using the methods of the present invention.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of the filing date of U.S.Provisional Application No. 60/493,322, filed Aug. 8, 2003, thedisclosure of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is in the fields of biotechnology and molecularbiology. More particularly, the present invention relates to seamlesslycloning or subcloning one or more nucleic acid molecules. The presentinvention also relates to seamless cloning of nucleic acid moleculescomprising one or more type IIs restriction enzyme recognition sites.The present invention also embodies cloning such nucleic acid moleculesusing recombinational cloning methods such as those employingrecombination sites and recombination proteins. The present inventionalso relates to nucleic acid molecules (including RNA and iRNA), as wellas proteins, expressed from host cells produced using the methods of thepresent invention.

2. Related Art

A significant problem with many of the currently available molecularcloning techniques results from the reliance upon restriction sites.These techniques result in the presence of extraneous polynucleotides inthe amplification products even after restriction digestions. Suchextraneous polynucleotides can introduce design limitations on thecloned product which often interfere with the structure and function ofthe desired gene products, be they RNA, DNA or protein.

One method of joining nucleic acids without introducing extraneous basesor relying on the presence of restriction sites is splice overlapextension (SOE) (Yon et al, Nucl. Acids Res. 17:4895 (1989) and Hortonet al., Gene 77:61-68 (1989)). This method is based on the hybridizationof homologous 3′ single-stranded overhangs to prime synthesis of DNAusing each complementary strand as a template. Although this techniquecan join fragments without introducing extraneous nucleotides (in otherwords, seamlessly), it does not permit the easy insertion of a DNAsegment into a specific location when seamless junctions at both ends ofthe segment are required. Nor does this technique allow for joiningfragments with a vector. Ligation with a vector must be subsequentlyperformed by incorporating restriction sites onto the termini of thefinal SOE fragment. Finally, this technique is particularly awkward whentrying to exchange polynucleotides encoding various domains or mutationsites between genetic constructs encoding related proteins.

Sorge et al., U.S. Pat. No. 6,261,797 describe a method by whichpolynucleotide sequences of interest are synthesized using one or moresynthesis primers, wherein at least one of the primers is a releasableprimer. After synthesis, the synthesis product is cleaved by a releasingenzyme. The releasable primers of Sorge et al. comprise a recognitionsite for a type IIs restriction endonuclease, principally Eam1105I. Thisthen allows for “seamless domain replacement” where synthesis reactionsallow the production of a polynucleotide of interest by synthesizing twodifferent polynucleotide sequences using separate sets of primers,cleaving the synthesis products with a releasing enzyme, and ligatingtogether the two sets of release synthesis products.

Type IIs Restriction Enzymes

Restriction enzymes can be grouped based on similar characteristics. Ingeneral there are three major types or classes: I, II (including IIs)and III. Class I enzymes cut at a somewhat random site from the enzymerecognition sites (see Old and Primrose, Principles of GeneManipulation, Blackwell Sciences, Inc., Cambridge, Mass., (1994)). Mostenzymes used in molecular biology are type II enzymes. These enzymesrecognize a particular target sequence (i.e., restriction endonucleaserecognition site) and break the polynucleotide chains within or near tothe recognition site. The type II recognition sequences are continuousor interrupted. Class IIs enzymes (i.e., type IIs enzymes) haveasymmetric recognition sequences. Cleavage occurs at a distance from therecognition site. These enzymes have been reviewed by Szybalski et al.Gene 100:13-26 (1991). Class III restriction enzymes are rare and arenot commonly used in molecular biology.

Type-IIs endonucleases generally recognize non-palindromic sequences andcleave outside of their recognition site, thus producing overhangs ofambiguous base pairs. (Szybalski, Gene 40:169-173 (1985).) Additionally,as a result of their non-palindromic recognition sequences, the use oftype-IIs endonucleases will generate more markers per kB than a similartype-II endonuclease, e.g., approximately twice as often. U.S. Pat. No.4,293,652 discloses a linker with a type-IIs enzyme recognition sequenceto permit synthesized DNA to be inserted into a vector withoutdisturbing a recognition sequence. Brousseau et al. (Gene 17:279-289(1982)) and Urdea et al. (Proc. Natl. Acad. Sci. USA 80:7461-7465(1983)) disclose the use of type-IIs enzymes for the production ofvectors to produce recombinant insulin and epidermal growth factorrespectively.

Thus, there remains a need in the art for methods and compositions thatallow for insertion of nucleic acid molecules into specific locations ofother nucleic acid molecules with seamless junctions at one or bothends. There is also a need in the art for methods and compositions thatallow for transfer of these seamlessly cloned sections from one nucleicacid molecule to another. The present invention fulfills these needs.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods of seamlessly cloning nucleicacid molecules. The seamless cloning methods of the present inventionmay utilize, for example, any restriction enzyme, including those whichcleave nucleic acid molecules to produce blunt ends. Suitably, themethods of the invention utilize type IIs restriction sites and enzymesthat recognize and cleave at such sites, which allow for the insertionof one or more (e.g. one, two, three, four, five, etc.) nucleic acidsegments into specific locations of a second nucleic acid molecule withseamless junctions on one or both ends. The present methods are alsosuitable for the production of nucleic acid molecules (e.g. DNA, RNA,DNA hybrids and the like) that only contain nucleic acid sequences thatare desired in the product molecule and that lack extraneous unwantedsequences, for example sequences comprising or encoded by restrictionsites. The present invention also provides for protein moleculesproduced or encoded by the cloned nucleic acid molecules of theinvention, that contain only amino acid sequences that are desired inthe product protein molecule (e.g., a native or mature protein, a fusionprotein, and the like), and that lack extraneous amino acids, forexample amino acids encoded by restriction sites. In certainembodiments, nucleic acid molecules of the present invention areespecially suitable for use as interfering RNA. The present inventionalso provides novel vectors comprising type IIs sites and, optionally,selectable markers for the production of seamlessly cloned nucleicacids, as well as compositions and kits for practicing methods of theinvention.

In one aspect, the present invention provides methods for joining one ormore (e.g. one, two, three, four, five, etc.) first nucleic acidmolecules and one or more second nucleic acid molecules, comprising: (a)combining the first and second nucleic acid molecules under conditionssufficient to allow for the joining of at least one terminus of thefirst nucleic acid molecule(s) to at least one terminus of the secondnucleic acid molecule(s), wherein the terminus of the first nucleic acidmolecule(s) which is connected to the terminus of the second nucleicacid molecule(s) comprises a sticky end (e.g. an overhanging end)generated by a restriction enzyme (e.g. a type IIs restriction enzyme)and the terminus of the second nucleic acid molecule(s) is compatible(e.g. a blunt end or a sticky end) with this sticky end. In embodimentssimilar to the above and elsewhere herein, the sticky end may be on theterminus of the second nucleic acid molecule, and the first nucleic acidmolecule may contain the compatible end.

In suitable such embodiments, the present invention provides methods ofcloning or subcloning one or more desired nucleic acid moleculescomprising: (a) combining in vitro or in vivo, (i) one or more firstnucleic acid molecules comprising one or more sticky ends that have beengenerated by one or more restriction enzymes (e.g. one or more type IIsrestriction enzymes); and (ii) one or more second nucleic acid moleculescomprising one or more ends which are compatible with the one or moresticky ends on the first nucleic acid molecule(s) and, optionally, oneor more selectable markers; and (b) incubating the combination underconditions sufficient to join the first nucleic acid molecule and one ormore of the second nucleic acid molecules, thereby producing one or moredesired product nucleic acid molecules.

In other aspects, the present invention provides methods for cloning orsubcloning one or more desired nucleic acid molecules comprising: (a)combining in vitro or in vivo, (i) one or more first nucleic acidmolecules comprising one or more sticky ends that have been generated byone or more restriction enzymes (e.g. one or more type IIs restrictionenzymes); (ii) one or more second nucleic acid molecules comprising oneor more restriction sites (e.g. one or more first type IIs restrictionenzyme recognition sites) and, optionally, one or more selectablemarkers; and (iii) one or more restriction enzymes (e.g., one or moretype IIs restriction enzymes) that are specific for the one or morerestriction sites on the second molecules; and (b) incubating thecombination under conditions sufficient to join the first nucleic acidmolecule and one or more of the second nucleic acid molecules, therebyproducing one or more desired product nucleic acid molecules.

In additional related aspects, the present invention provides methodsfor cloning or subcloning one or more desired nucleic acid moleculescomprising: (a) combining in vitro or in vivo, (i) one or more firstnucleic acid molecules comprising at least one nucleic acid segment thatis flanked by one or more restriction sites (e.g. one or more first typeIIs restriction enzyme recognition sites); (ii) one or more secondnucleic acid molecules comprising one or more ends which are compatiblewith a sticky end on the segment and, optionally, one or more selectablemarkers; and (iii) one or more restriction enzymes (e.g., one or moretype IIs restriction enzymes) that are specific for the one or morerestriction sites on the at least one nucleic acid segment; and (b)incubating the combination under conditions sufficient to join the firstnucleic acid segment and one or more of the second nucleic acidmolecules, thereby producing one or more desired product nucleic acidmolecules.

In related aspects, the present invention provides methods for cloningor subcloning one or more desired nucleic acid molecules, or portionsthereof, comprising: (a) combining in vitro or in vivo, (i) one or morefirst nucleic acid molecules comprising at least one nucleic acidsegment that is flanked by one or more first restriction sites (e.g. oneor more first type IIs restriction enzyme recognition sites); (ii) oneor more second nucleic acid molecules comprising one or more secondrestriction sites (e.g. one or more type IIs restriction enzymerecognition sites) and, optionally, one or more selectable markers; and(iii) one or more restriction enzymes (e.g. one or more type IIsrestriction enzymes) that are specific for the first and/or second typeIIs restriction enzyme recognition sites; and (b) incubating thecombination under conditions sufficient to join the first nucleic acidsegment and one or more of the second nucleic acid molecules, therebyproducing one or more desired product nucleic acid molecules.

Type IIs restriction enzyme recognition sites and type IIs restrictionenzymes that are useful in the present cloning methods, compositions,nucleic acids, vectors and kits include, but are not limited to, BsaI,BbsI, BbvII, BsmAI, BspMI, Eco31I, BsmBI, BaeI, FokI, HgaI, MlyI, SfaNIand Sth132I. The first, and second restriction sites, if present,utilized throughout the various aspects of the present invention may bethe same or they may be different. In addition, the restriction sites onthe same nucleic acid molecule (and/or nucleic acid segment) may be thesame, or they may be different. The present invention also encompassessituations wherein one or both of the nucleic acid molecules involved inthe various methods are vectors, and where one or both of the nucleicacid molecules are linear nucleic acid molecules. The present inventionalso encompasses the use of other blunt-end cleavage enzymes, including,but not limited to, ScaI, SmaI, HpaI, HincII, HaeII and AluI.

In certain embodiments, the nucleic acids and nucleic acid segmentsutilized in the cloning methods, compositions, kits, and vectors of thepresent invention may optionally comprise one or more selectablemarkers. Hence, the invention also provides such nucleic acids. The oneor more selectable markers utilized in the present invention may beflanked by one or more (e.g. one, two, three, four, five, etc.)restriction sites (e.g. type IIs restriction enzyme recognition sites).Suitable selectable markers include, but are not limited to, genes thatconfer antibiotic resistance, genes that encode fluorescent proteins,tRNA genes, auxotrophic markers, toxic genes, phenotypic markers,antisense oligonucleotides, restriction endonucleases, restrictionendonuclease cleavage sites, enzyme cleavage sites, protein bindingsites, and sequences complementary to PCR primer sequences. Suitableantibiotic resistance genes include, but are not limited to, achloramphenicol resistance gene, an ampicillin resistance gene, atetracycline resistance gene, a Zeocin resistance gene, a spectinomycinresistance gene and a kanamycin resistance gene. In certain embodimentsof the present invention, the selectable marker is a toxic gene.Suitable toxic genes include, but are not limited to, a ccdB gene, agene encoding a tus protein which binds one or more ter sites, a kicBgene, a sacB gene, an ASK1 gene, a ΦX174 E gene and a DpnI gene. Inadditional embodiments of the methods of the present invention, thefirst and/or second nucleic acid molecules may comprise both one or moretoxic genes and one or more antibiotic resistance genes, and these genesmay further be flanked by type IIs restriction enzyme recognition sites.In suitable such embodiments of the present invention, the first and/orsecond nucleic acid molecules may comprise both a toxic gene and anantibiotic resistance gene.

In other aspects of the invention, nucleic acids and/or nucleic acidsegments for use in the cloning methods, vectors, kits and compositionsmay further comprise one or more recombination sites and/or one or moretopoisomerase recognition sites and/or one or more topoisomerases. Thenucleic acids and/or nucleic acid segments of the present invention mayalso comprise two or more recombination sites. If a topoisomeraserecognition site is present in a nucleic acid molecule or nucleic acidsegment of the present invention, it may optionally be flanked by two ormore recombination sites. Recombination sites suitable for use in thepresent invention include, but are not limited to, attB sites, attPsites, attL sites, attR sites, lox sites, psi sites, tnpI sites, difsites, cer sites, frt sites, and mutants, variants and derivativesthereof. These one or more recombination sites may flank one or moreselectable markers, if present, and/or restriction sites (e.g. type IIssites). In certain embodiments of the present invention, thetopoisomerase recognition site, if present, is recognized and bound by atype I topoisomerase, which may be a type IB topoisomerase. Suitabletypes of type IB topoisomerase include, but are not limited to,eukaryotic nuclear type I topoisomerase and poxvirus topoisomerase.Suitable types of poxvirus topoisomerase include, but are not limitedto, poxvirus topoisomerase produced by or isolated from a virus such asvaccinia virus, Shope fibroma virus, ORF virus, fowlpox virus, molluscumcontagiosum virus and Amsacta morrei entomopoxvirus.

The present invention also provides methods of linking nucleic acidmolecules and/or nucleic acid segments which comprise one or moretopoisomerases bound to one or both termini, wherein the topoisomeraseadapted terminus or termini comprise a sequence compatible with thatcleaved by a restriction enzyme (e.g. a type IIs restriction enzyme). Insuch suitable embodiments of the invention, a first nucleic acidmolecule or nucleic acid segment may contain a blunt end to be linked,and a second nucleic acid molecule may contain an overhang at the endwhich is to be linked by a site-specific topoisomerase (e.g., a type IAor a type IB topoisomerase), wherein the overhang includes a sequencecomplementary to that comprising the blunt end, thereby facilitatingstrand invasion as a means to properly position the ends for the linkingreaction.

The nucleic acid molecules generated using this aspect of the inventioninclude those in which at least one strand (not both strands) iscovalently linked at the ends which are joined (e.g. double-strandednucleic acid molecules generated using these methods contain a nick ateach position where two ends were joined). These embodiments areparticularly advantageous in that a polymerase can be used to replicatethe double-stranded (ds) nucleic acid molecule by initially replicatingthe covalently linked strand. For example, a thermostable polymerasesuch as a polymerase useful for performing an amplification reactionsuch as PCR can be used to replicate the covalently strand, whereas thestrand containing the nick does not provide a suitable template forreplication.

In certain embodiments of the invention, the first or second nucleicacid molecules or nucleic acid segments involved in the various methodsof the present invention may not comprise a promoter. The presentinvention also allows for transfer of a promoter element into a secondnucleic acid molecule that may not comprise a promoter, via seamlesscloning. In this orientation, transcription of the second nucleic acidmolecule from the promoter element located on the first nucleic acidmolecule or nucleic acid segment may proceed such that no additionalsequences are transcribed between the promoter element and thetranscription initiation point of the second nucleic acid molecule. Thepresent invention also allows for seamlessly adding a first nucleic acidmolecule or nucleic acid segment into a second nucleic molecule thatcontains a promoter element such that the first nucleic acid molecule orsegment will subsequently be under the control of the promoter element.

The present invention also provides methods for cloning or subcloningone or more desired nucleic acids: (a) combining in vitro or in vivo,(i) one or more first nucleic acid molecules that have one or moresticky ends that have been generated by one or more restriction enzymes(e.g. type IIs restriction enzymes); and (ii) one or more second nucleicacid molecules comprising one or more ends which are compatible with theone or more sticky ends on the first nucleic acid molecule(s) andfurther comprising one or more recombination sites; and (b) incubatingthe combination under conditions sufficient to join the first nucleicacid molecule and one or more of the second nucleic acid molecules,thereby producing one or more desired product nucleic acid molecules.

The present invention also provides methods for cloning or subcloningone or more desired nucleic acid molecules, or portions thereof,comprising: (a) combining in vitro or in vivo, (i) one or more firstnucleic acid molecules comprising at least one nucleic acid segment thatis flanked by one or more first restriction sites (e.g. one or more typeIIs restriction enzyme recognition sites); (ii) one or more secondnucleic acid molecules comprising one or more second restriction sites(e.g. type IIs restriction enzyme recognition sites) flanked by one ormore recombination sites; and (iii) one or more restriction enzymes(e.g. one or more type IIs restriction enzymes) that are specific forthe first and/or second restriction sites; and (b) incubating thecombination under conditions sufficient to join the first nucleic acidmolecule and one or more of the second nucleic acid molecules, therebyproducing one or more desired product nucleic acid molecules.

As described above, the first and/or second nucleic acid moleculesand/or nucleic acid segments involved in such embodiments of the presentinvention may optionally comprise one or more selectable markers. Thefirst and/or second nucleic acid molecules and/or nucleic acid segmentsinvolved in such aspects of the invention may also, or alternativelycomprise one or more topoisomerase recognition sites or topoisomerasesas described above, and optionally or alternatively, two or morerecombination sites, which in certain such embodiments may flank thesetopoisomerases or topoisomerase recognition sites.

The present invention also provides methods for cloning or subcloningone or more desired nucleic acid molecules, or portions thereof, viarecombination cloning comprising: (a) combining, in vitro or in vivo (i)one or more first nucleic acid molecules comprising at least one nucleicacid segment that is flanked by one or more restriction sites (e.g. oneor more type IIs restriction enzyme recognition sites) and that isfurther flanked by one or more recombination sites; (ii) one or moresecond nucleic acid molecules comprising one or more recombinationsites; and (iii) one or more site-specific recombination proteins; and(b) incubating the combination under conditions sufficient to join thefirst nucleic acid molecule and one or more of the second nucleic acidmolecules, thereby producing one or more desired product nucleic acidmolecules.

The second nucleic acid molecule involved in such embodiments of theinvention may also comprise one or more restriction sites (e.g. one ormore type IIs restriction enzyme recognition sites). The first and/orsecond nucleic acids and/or nucleic acid segments involved may alsooptionally comprise one or more selectable markers as described above.The first and/or second nucleic acid molecules and/or nucleic acidsegments involved in this aspect of the invention may also comprisetopoisomerase recognition sites or topoisomerases as described above, aswell as two or more recombination sites flanking these topoisomerasesites.

Suitable recombination proteins for use in the present inventioninclude, but are not limited to, Int, Cre, IHF, Xis, Fis, Hin, Gin, Cin,Tn3 resolvase, TndX, XerC and XerD.

The present invention also provides methods for producing host cellscomprising one or more of the nucleic acid molecules produced by thecloning methods of the present invention Suitable host cells that may beused throughout the present invention include, but are not limited to,bacterial cells, yeast cells, plant cells and animal cells. The presentinvention also provides methods for producing a subsequent nucleic acidmolecule and/or protein by expression of the product nucleic acidmolecule of the cloning methods of the present invention in a host cell.

Additional embodiments provide for nucleic acid molecules and proteinsproduced in and isolated from a host cell. In certain such embodiments,the nucleic acid molecules produced in the host cell may contain onlydesired nucleic acid sequences, i.e. they may not contain extraneousnucleotides, for example, nucleotides encoded by the restriction sites(e.g. type IIs restriction enzyme recognition sites). Similarly, theproteins produced from a host cell by these methods may only containamino acid sequences that correspond to the desired native or matureprotein, and may not contain extraneous amino acids, for example aminoacids encoded by the restriction sites (e.g. type IIs restriction enzymerecognition sites). Nucleic acid molecules produced from a host cell bymethods of the present invention may be useful as interfering RNAmolecules.

Another aspect of the present invention provides methods of producing anRNA molecule for use as an interfering RNA comprising: (a) optionally,identifying one or more target nucleic acid sequences; (b) preparing oneor more nucleic acid molecules which encode one or more interferingRNAs, wherein the interfering RNAs bind to the one or more targetnucleic acid sequences; (c) combining in vitro or in vivo, (i) the oneor more first nucleic acid molecules encoding one or more interferingRNAs that have one or more sticky ends that have been generated by oneor more restriction enzymes (e.g. type IIs restriction enzymes); and(ii) one or more second nucleic acid molecules comprising one or moreends which are compatible with the one or more sticky ends on the firstnucleic acid molecule(s), and optionally comprising one or moreselectable markers; and (d) incubating the combination under conditionssufficient to join one or more of the nucleic acid molecules encodingthe interfering RNAs and one or more of the second nucleic acidmolecules, thereby producing one or more desired product nucleic acidmolecules; (e) inserting the one or more product nucleic acid moleculesinto a host cell; and (f) expressing the one or more interfering RNAs inthe host cell. As in other embodiments of the invention describedherein, the second nucleic acid molecule may contain an end which isgenerated by digestion with a type IIs restriction enzyme and the firstnucleic acid-molecule may contain a compatible end generated by othermeans.

The present invention also provides methods of producing an RNA moleculefor use as an interfering RNA comprising: (a) optionally, identifyingone or more target nucleic acid sequences; (b) preparing one or morenucleic acid molecules which encode one or more interfering RNAs,wherein the interfering RNAs bind to the one or more target nucleic acidsequences; (c) combining in vitro or in vivo, (i) the one or more firstnucleic acid molecules encoding one or more interfering RNAs flanked byone or more first restriction sites (e.g. one or more type IIsrestriction enzyme recognition sites); (ii) one or more second nucleicacid molecules comprising one or more second restriction sites (e.g. oneor more type IIs restriction enzyme recognition sites) and optionallycomprising one or more selectable markers; and (iii) one or moresite-specific restriction enzymes (e.g. one or more type IIs restrictionenzymes); and (d) incubating the combination under conditions sufficientto join one or more of the nucleic acid molecules encoding theinterfering RNAs and one or more of the second nucleic acid molecules,thereby producing one or more desired product nucleic acid molecules;(e) inserting the one or more product nucleic acid molecules into a hostcell; and (f) expressing the one or more interfering RNAs in the hostcell.

In related embodiments, the present invention provides methods ofproducing an RNA molecule for use as an interfering RNA comprising: (a)optionally, identifying one or more target nucleic acid sequences; (b)preparing one or more nucleic acid molecules which encode one or moreinterfering RNAs, wherein the interfering RNAs bind to the one or moretarget nucleic acid sequences; (c) combining in vitro or in vivo, (i)the one or more first nucleic acid molecules encoding one or moreinterfering RNAs that have one or more sticky ends that have beengenerated by one or more restriction enzymes (e.g. type IIs restrictionenzymes); and (ii) one or more second nucleic acid molecules comprisingone or more ends which are compatible with the one or more sticky endson the first nucleic acid molecule(s), and optionally comprising one ormore selectable markers; and (d) incubating the combination underconditions sufficient to join one or more of the nucleic acid moleculesencoding the interfering RNAs and one or more of the second nucleic acidmolecules, thereby producing one or more desired product nucleic acidmolecules; and (e) expressing one or more interfering RNAs in vitro orin vivo. In a first further embodiment, the one or more interfering RNAsmay be produced in vitro or isolaged from a cell and then introducedinto a second cell.

Another aspect of the present invention provides methods of producing anRNA molecule for use as an interfering RNA comprising: (a) optionally,identifying one or more target nucleic acid sequences; (b) preparing oneor more nucleic acid molecules which encode one or more interferingRNAs, wherein the interfering RNAs bind to the one or more targetnucleic acid sequences; (c) combining in vitro or in vivo, (i) the oneor more first nucleic acid molecules encoding one or more interferingRNAs flanked by one or more first restriction sites (e.g. one or moretype IIs restriction enzyme recognition sites); (ii) one or more secondnucleic acid molecules comprising one or more second restriction sites(e.g. one or more type IIs restriction enzyme recognition sites) andoptionally comprising one or more selectable markers; and (iii) one ormore site-specific restriction enzymes (e.g. one or more type IIsrestriction enzymes); and (d) incubating the combination underconditions sufficient to join one or more of the nucleic acid moleculesencoding the interfering RNAs and one or more of the second nucleic acidmolecules, thereby producing one or more desired product nucleic acidmolecules; and (e) expressing one or more interfering RNAs in vitro orin vivo. In a first further embodiment, the one or more interfering RNAsmay be produced in vitro or isolaged from a cell and then introducedinto a second cell.

In a related aspect, the present invention provides methods of producingan RNA molecule for use as an interfering RNA comprising: (a)optionally, identifying one or more target nucleic acid sequences; (b)preparing one or more interfering RNAs, wherein the interfering RNAsbind to the one or more target nucleic acid sequences; (c) combining invitro or in vivo, (i) the one or more first nucleic acid moleculescomprising one or more interfering RNAs that have one or more stickyends that have been generated by one or more restriction enzymes (e.g.type IIs restriction enzymes); and (ii) one or more second nucleic acidmolecules comprising one or more ends which are compatible with the oneor more sticky ends on the first nucleic acid molecule(s), andoptionally comprising one or more selectable markers; and (d) incubatingthe combination under conditions sufficient to join one or moreinterfering RNAs and one or more of the second nucleic acid molecules,thereby producing one or more desired product nucleic acid molecules;(e) inserting the one or more product nucleic acid molecules into a hostcell; and (f) expressing the one or more interfering RNAs in the hostcell.

The present invention also provides methods of producing an RNA moleculefor use as an interfering RNA comprising: (a) optionally, identifyingone or more target nucleic acid sequences; (b) preparing one or morenucleic acid molecules which comprise one or more interfering RNAs,wherein the interfering RNAs bind to the one or more target nucleic acidsequences; (c) combining in vitro or in vivo, (i) the one or more firstnucleic acid molecules comprising one or more interfering RNAs flankedby one or more first restriction sites (e.g. one or more type IIsrestriction enzyme recognition sites); (ii) one or more second nucleicacid molecules comprising one or more second restriction sites (e.g. oneor more type IIs restriction enzyme recognition sites) and optionallycomprising one or more selectable markers; and (iii) one or moresite-specific restriction enzymes (e.g. one or more type IIs restrictionenzymes); and (d) incubating the combination under conditions sufficientto join one or more interfering RNAs and one or more of the secondnucleic acid molecules, thereby producing one or more desired productnucleic acid molecules; (e) inserting the one or more product nucleicacid molecules into a host cell; and (f) expressing the one or moreinterfering RNAs in the host cell.

Methods of the present invention may be used, for example, to prepareshRNA molecules in which the 5′ and 3′ termini contain none or few(e.g., one, two, three, four, or five) nucleotides which are not encodedby a first nucleic acid molecule referred to throughout. Thus, the shRNAmay comprise from about 40 to about 60 nucleotides in which either noneof all but a few nucleotides at one or both termini are encoded by afirst nucleic acid molecule. In such instances, the first nucleic acidmolecule may be composed of nucleic acid which upon transcriptionresults in the production of RNA with three different segments: (1)sense RNA, (2) a loop/non-complementary RNA, and (3) antisense RNA.Methods of the invention include introducing into a cell (1)(a) nucleicacid which encodes the RNA described above or (b) the RNA itself, and(2) the measurement of inhibition of expression of a gene correspondingto the sense and/or antisense RNA.

In particular embodiments of the invention, the invention may be used toproduce nucleic acid molecules which produce RNA molecules that do notform hairpins. As one example, methods of the invention may be used toproduce two separate vectors, one or which may be used to produce asense RNA molecules (e.g., a sense RNA molecule which is between about18 and about 30, between about 20 and about 30, between about 22 andabout 30, or between about 18 and about 25 nucleotides in length) and anantisense RNA molecules (e.g., a sense RNA molecule which is betweenabout 18 and about 30, between about 20 and about 30, between about 22and about 30, between about 18 and about 100, or between about 18 andabout 25 nucleotides in length), wherein the two RNA molecules arecapable of hybridizing to each other and/or share a region of sequencecomplementarity over at least 80%, 90%, or 95% of their full lengths(e.g., sequence complementarity over a 19 nucleotide stretch, whereineach molecule is 22 nucleotides in length). Alternatively, both senseand antisense RNA molecules, such as described above, may be produced bya single vector but as separate transcription products.

As a variation of the above, the invention may be used to produce eithersense or antisense RNA molecules alone in cells. These RNA molecules maybe of any length suitable for the particular application (e.g.,expression of protein, antisense inhibition of gene expression, ribozymeproduction, etc.).

The invention may further be used to produce microRNA molecules.MicroRNA molecules are molecules which are structurally similar to shRNAmolecules but, typically, contain one or more mismatches orinsertion/deletions in their regions of sequence complementary. At leastsome microRNA molecules are transcribed as polycistrons of about 400,which are then processed to RNA molecules of about 70 nucleotides. Thesedouble stranded 70 mers are then are processed again, presumably by theenzyme Dicer, to two RNA molecules which are about 22 nucleotides inlength and often have one or more (e.g., one, two, three, four, five,etc.) internal mismatches in their regions of sequence complementarity.Lee et al., EMBO 21:4663-4670 (2002). The invention also includes, forexample, uses of microRNA molecules and nucleic acid molecules whichencode microRNA molecules which are similar to the uses described thosedescribed herein for shRNA and non-hairpin doule stranded RNA molecules.

The present invention also provides methods of regulating the expressionof one or more genes in a cell or an animal using interfering RNA,comprising: (a) identifying one or more target nucleic acid sequences;(b) preparing one or more nucleic acid molecules which encode one ormore interfering RNAs, wherein the interfering RNAs bind to the one ormore target nucleic acid sequences; (c) combining in vitro or in vivo,(i) the one or more first nucleic acid molecules encoding one or moreinterfering RNAs that have one or more sticky ends that have beengenerated by one or more restriction enzymes (e.g. type IIs restrictionenzymes); and (ii) one or more second nucleic acid molecules comprisingone or more ends which are compatible with the one or more sticky endson the first nucleic acid molecule(s), and optionally comprising one ormore selectable markers; (d) incubating the combination under conditionssufficient to join one or more of the nucleic acid molecules encodingthe interfering RNAs and one or more of the second nucleic acidmolecules, thereby producing one or more desired product nucleic acidmolecules; and (e) inserting the one or more interfering RNA expressionvectors into the cell or one or more cells of the animal, underconditions such that the one or more interfering RNAs bind to the one ormore target nucleic acid sequences, thereby regulating expression of theone or more targeted genes.

In related embodiments, the present invention also provides methods ofregulating the expression of one or more genes in a cell or an animalusing interfering RNA, comprising: (a) identifying one or more targetnucleic acid sequences; (b) preparing one or more nucleic acid moleculeswhich comprise one or more interfering RNAs, wherein the interferingRNAs bind to the one or more target nucleic acid sequences; (c)combining in vitro or in vivo, (i) the one or more first nucleic acidmolecules comprising one or more interfering RNAs flanked by one or morefirst restriction sites (e.g. one or more type IIs restriction enzymerecognition sites); (ii) one or more second nucleic acid moleculescomprising one or more second restriction sites (e.g. one or more typeIIs restriction enzyme recognition sites) and optionally comprising oneor more selectable markers; and (iii) one or more site-specificrestriction enzymes (e.g. one or more type IIs restriction enzymes); (d)incubating the combination under conditions sufficient to join one ormore interfering RNAs and one or more of the second nucleic acidmolecules, thereby producing one or more desired product nucleic acidmolecules; and (e) inserting the one or more interfering RNA expressionvectors into the cell or one or more cells of the animal, underconditions such that the one or more interfering RNAs bind to the one ormore target nucleic acid sequences, thereby regulating expression of theone or more targeted genes.

Such methods of the invention can be used to knockout or knockdown oneor more genes in vivo in a cell or animal. These methods of theinvention may also be used to produce genetically modified animals byexpressing interfering RNA in germ cells or somatic cells, and forpreparation of transgenic animals.

In another embodiment, the present invention also provides isolatednucleic acid molecules comprising: (a) one or more sticky ends that havebeen generated by one or more restriction enzymes (e.g. one or more typeIIs restriction enzymes); and (b) optionally one or more selectablemarkers. The present invention also provides isolated nucleic acidmolecules comprising: (a) one or more restriction sites (e.g. one ormore type IIs restriction enzyme recognition sites); and (b) optionallyone or more selectable markers.

Suitable restriction enzyme recognition sites and selectable markers aredescribed above. The isolated nucleic acid molecules of the presentinvention may also comprise one or more recombination sites and/or oneor more topoisomerase recognition sites and/or one or moretopoisomerases. If present, the topoisomerase recognition sites may beflanked by recombination sites. The isolated nucleic acid molecules ofthe present invention may be vectors or linear nucleic acid molecules.The present invention also provides isolated nucleic acid moleculescomprising: (a) one or more sticky ends that have been generated by oneor more restriction enzymes (e.g. one or more type IIs restrictionenzymes); and (b) one or more recombination sites. The present inventionfurther provides isolated nucleic acid molecules comprising: (a) one ormore restriction sites (e.g. one or more type IIs restriction enzymerecognition sites); and (b) one or more recombination sites.

The present invention also provides vectors comprising: (a) one or moredesired nucleic acid segments; (b) optionally one or more toxic genes;and (c) one or more sites that are compatible with a sticky endgenerated by a restriction enzyme (e.g. one or more type IIs restrictionenzymes). Suitable desired nucleic acid molecules include genes (e.g.open reading frames) and promoters. The vectors of the present inventionmay also comprise one or more recombination sites, and one or moretopoisomerase recognition sites and/or one or more topoisomerases,wherein, the topoisomerase recognition sites if present, may be flankedby recombination sites. In other embodiments, the vectors of the presentinvention may optionally comprise one or more selectable markers asdescribed above. Suitable vectors of the present invention include, butare not limited to, pENTR/U6-ccdB (vector diagram for pENTR/U6-ccdBshown in FIG. 2A, vector sequence in Table 5, FIG. 12, and SEQ ID NO:1).

The present invention also provides vectors comprising: (a) one or moredesired nucleic acid segments; (b) optionally one or more toxic genes;and (c) one or more restriction sites (e.g. one or more type IIsrestriction enzyme recognition sites). Suitable desired nucleic acidmolecules include genes and promoters. The vectors of the presentinvention may also comprise one or more recombination sites, and one ormore topoisomerase recognition sites and/or one or more topoisomerases,wherein, the topoisomerase recognition sites if present, may be flankedby recombination sites. In other embodiments, the vectors of the presentinvention may optionally comprise one or more selectable markers asdescribed above. Suitable vectors of the present invention include, butare not limited to, pENTR/U6-ccdB (vector diagram for pENTR/U6-ccdBshown in FIG. 2A, vector sequence in Table 5, FIG. 12, and SEQ ID NO:1).

The present invention also provides host cells comprising one or more ofthe isolated nucleic acid molecules or nucleic acid segments of thepresent invention, and methods of expressing the isolated nucleic acidsof the present invention in one more host cells and isolating theexpressed nucleic acids. The present invention also provides methods ofexpressing and isolating proteins from host cells comprising one or moreisolated nucleic acids or nucleic acid segments of the invention.

Another embodiment of the invention provides methods of expressingdesired product nucleic acid segments by introducing the nucleic acidmolecules, nucleic acid segments, or vectors of the present inventioninto a host cell and expressing the product nucleic acid segments.

The present invention also provides for compositions comprising: (a) oneor more first nucleic acid molecules that have one or more sticky endsthat have been generated by one or more restriction enzymes (e.g. typeIIs restriction enzymes); and (ii) one or more second nucleic acidmolecules comprising one or more ends which are compatible with the oneor more sticky ends on the first nucleic acid molecule(s). The first andsecond nucleic acid molecules may optionally comprise one or moreselectable markers as discussed above. These first and second nucleicacid molecules may also comprise one or more recombination sites, one ormore topoisomerase recognition sites and/or one or topoisomerases,wherein the topoisomerase recognition sites, if present, may be flankedby recombination sites. The optional selectable markers may be flankedby type IIs restriction sites and/or recombination sites. Thecompositions of the invention may also comprise one or morerecombination proteins as described above.

The present invention further provides for compositions comprising: (a)one or more first nucleic acid molecules comprising at least one nucleicacid segment that is flanked by one or more first restriction sites(e.g. one or more type IIs restriction enzyme recognition sites; (b) oneor more second nucleic acid molecules optionally comprising one or moresecond restriction sites (e.g. one or more type IIs restriction enzymerecognition sites); and (c) one or more restriction enzymes (e.g. typeIIs restriction enzymes) that are specific for the first and/or secondrestriction sites. The first and second nucleic acid molecules and/ornucleic acid segments may optionally comprise one or more selectablemarkers as discussed above. These first and second nucleic acidmolecules and/or nucleic acid segments may also comprise one or morerecombination sites, one or more topoisomerase recognition sites and/orone or topoisomerases, wherein the topoisomerase recognition sites, ifpresent, may be flanked by recombination sites. The optional selectablemarkers may be flanked by type IIs restriction sites and/orrecombination sites. The compositions of the invention may also compriseone or more recombination proteins as described above.

The present invention also provides kits comprising the isolated nucleicacids or vectors of the present invention. The kits of the presentinvention may further comprise one or more type IIs restriction enzymes,one or more recombination proteins, and one or more host cells.

Other embodiments of the present invention will be apparent to one orordinary skill in light of the following drawings and description of theinvention, and of the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic diagram of a vector of the invention comprising:an origin of replication (ori), a kanamycin resistance gen (kan), aPolymerase II promoter (polII), L1 (attL1) and L2 (attL2) recombinationsites, an ATG translation initiation site/codon, a secretion signal,type IIs restriction sites, and a negative selectable marker.

FIG. 1B is a schematic diagram of a vector of the invention comprising:an origin of replication (ori), a kanamycin resistance gen (kan), aPolymerase II promoter (polII), L1 (attL1) and L2 (attL2) recombinationsites, an ATG initiation site/codon, an affinity tag, a cleavage site, atype IIs restriction site, and a negative selectable marker.

FIG. 2A is a schematic diagram of pENTR/U6.

FIG. 2B depicts a BsaI digestion and cloning scheme using pENTR/U6.

FIGS. 3A and 3B depict luciferase and β-gal suppression in GripTite™ 293cells by transient cotransfection of reporters and pENTR/U6 vectors. A)Luciferase activities measured in lysates of cells: from left 1)untransfected, 2) cotransfected with luciferase and lacZ reporter genesplus a dummy plasmid (pUC19/actin), or 3-4) same as 2 except eitherpENTR/U6 targeting luciferase (GL2-22) or β-gal (lacZ-19) replace thepUC19/actin. B) β-gal activity measurements of the same lysates as in A.Activities are the average of duplicate wells. The standard error of themean is indicated for each sample.

FIGS. 4A and 4B depicts RNAi of β-Gal and Luciferase activity fromco-transfected reporter constructs by pENTR/U6 shRNA clones. Data arereported as the ratio of lacZ and Luciferase activity. Error bars arecalculated from two independent samples. AS/SA indicates the orientationof the sense and anti-sense strand relative to the U6 promoter. A)Luciferase/β-gal activity after co-transfection with the indicatedpENTR/U6 shRNA sequences targeting the Luciferase gene and a pUC19-actincontrol. pENTR/U6-A6-GL2-22(AS) is the same construct used in FIG. 3.The asterisk (*) after ENTR/U6-A6-GL2-2-SA indicates a point mutationwas identified in the shRNA target sequence clone used in thisexperiment. B) β-gal/Luciferase activity after co-transfection withvarious pENTR/U6 shRNA sequences targeting the LacZ gene.ENTR/U6-A6-lacZ-19 is the same construct used to generate the datapresented in FIG. 3.

FIG. 5 depicts β-gal/Luciferase activity ratios after co-transfectionreporter plasmids and pENTR/U6 LacZ-19 shRNA target clones with theindicated Terminator lengths. Terminators with 4, 5, 6 and 8 “Ts” weretested in the pENTR/U6.2 vector (A4-8).

FIG. 6A is a schematic of the lentiviral RNAi shRNA transfer vector:pLenti6/RNAi-DEST which is a promoterless Gateway-adapted lenti vectorwhich may be used to clone, for example an shRNA cassette of interestvia Gateway L×R reaction with pENTR U6 vectors. The shRNA cassette willoften contain an RNA pol III- or other-promoter of choice to drivehairpin expression. The vector confers blasticidin resistance totransduced cells.

FIG. 6B is a schematic of the lentiviral RNAi Kit control vector: Kitcontrol plasmid pLenti6/RNAi/U6-GW/lamAC which results from L×R reactionbetween pLenti6/RNAi-DEST and pENTR/U6-lamAC-AS-cgaa.pLenti6/RNAi/U6-GW/lamAC expresses lamAC-AS-cgaa hairpin to specificallyknockdown lamin A/C expression.

FIG. 7 depicts the inhibition of lamin A/C expression. Lenti6/RNAiviruses encoding anti-lamin A/C shRNAs (U6-lamAC) were transduced intoHeLa cells to test inhibition of lamin A/C expression. Control virusesencoded GFP gene (GFP) or anti-luciferase shRNAs (U6-GL2). Western blotsfor lamin A/C or beta-actin were conducted on lysates from transducedcells. Top panel: Lysates were prepared 48 hrs post-transduction. Bottompanel: Lysates were prepared from transduced, shRNA-producing,blasticidin-resistant cells 5 days post-transduction.

FIG. 8A is a plasmid map of pLenti6/V5-DEST.

FIG. 8B is a plasmid map of pLenti6/V5-gTOPO®.

FIG. 8C is a plasmid map of pLenti4/V5-DEST

FIG. 8D is a plasmid map of pLenti6/UbC/V5-DEST.

FIG. 9A is a plasmid map pLP1.

FIG. 9B is a plasmid pLP2.

FIG. 9C is a plasmid map of pLP/VSVG.

FIG. 10 is a plasmid map of pAd/PL-DEST.

FIG. 11 is a plasmid map of pAd/CMV/V5-DEST.

FIG. 12 depicts the nucleic acid sequence of the pENTR/U6 withannotations noting the various segments of the vector. SEQ ID NO:1

FIG. 13 depicts RNAi overview.

FIG. 14 depicts RNAi Mechanistic Model.

FIG. 15 depicts RNAi Methods.

FIG. 16 depicts siRNA Molecules.

FIG. 17 depicts Transfection of siRNAs

FIG. 18 depicts Variation in siRNA effectiveness.

FIG. 19 depicts expression in vivo.

FIG. 20 depicts BLOCK-iT™ Long RNAi Transcription Kit.

FIG. 21 depicts BLOCK-iT™ Dicer RNAi Kit

FIG. 22 depicts d-siRNA knockdown.

FIG. 23 depicts d-siRNA vs. siRNA.

FIG. 24 depicts BLOCK-iT™ RNAi.

FIG. 25 depicts Micro RNA (miRNA).

FIG. 26 depicts RNAi Vectors.

FIG. 27 depicts U6 RNAi.

FIG. 28 depicts Gateway™ Cloning and ViraPower™ RNAI cassettes.

FIG. 29 depicts Selecting a viral expression system.

FIG. 30 depicts Outline for lentiviral production.

FIG. 31 depicts Overview of Lentiviral Production.

FIG. 32 depicts ViraPower™ lentiviral production.

FIG. 33 depicts Clone your gene of interest into Lentivirus.

FIG. 34 depicts Two methods for fast cloning.

FIG. 35 depicts Two methods for fast cloning.

FIG. 36 depicts Subcloning an Entry Clone into Multiple DestinationVectors.

FIG. 37 depicts pLenti6/V5 Expression Vectors.

FIG. 38 depicts GATEWAY Cloning Technology.

FIG. 39 depicts Assembly of Three DNA segments using Existing EntryClones.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs.

One or more: As used herein, the term “one or more” includes at leastone, more suitably, one, two, three, four, five, ten, twenty, fifty,one-hundred, five-hundred, etc., of the item to which “one or more”refers.

Nucleic Acid: As used herein, “nucleic acid” refers to polynucleotidessuch as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The termshould also be understood to include, as applicable to the embodimentbeing described, single-stranded (such as sense or antisense) and doublestranded polynucleotides, including double-stranded DNA-RNA hybrids. Theterm “nucleic acid” also is synonymous, and may be used interchangeablywith the term “nucleic acid molecule.”

Gene: As used herein, “gene” refers to a nucleic acid comprising an openreading frame encoding a polypeptide, including both exon and(optionally) intron sequences.

About: As used herein, when referring to any numerical value, “about”means a value of ±10% of the stated value (e.g. “about 50° C.encompasses a range of temperatures from 45° C. to 55° C., inclusive:similarly, “about 100 mM” encompasses a range of concentrations from 90mM to 110 mM, inclusive).

Host: As used herein, a “host” is any prokaryotic or eukaryotic organismthat is a recipient of a replicable expression vector, cloning vector orany nucleic acid molecule. The nucleic acid molecule may contain, but isnot limited to, a structural gene, a transcriptional regulatory sequence(such as a promoter, enhancer, repressor, and the like) and/or an originof replication. As used herein, the terms “host,” “host cell,”“recombinant host” and “recombinant host cell” may be used equivalentlyand interchangeably. For examples of such hosts, see Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1982).

Derivative: As used herein the term “derivative,” when used in referenceto a vector, means that the derivative vector contains one or more(e.g., one, two, three, four five, etc.) nucleic acid segments whichshare sequence similar to the vectors represented in FIG. 1A, FIG. 1B,FIG. 2A, FIG. 6A, FIG. 6B, FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D, FIG. 9A,FIG. 9B, FIG. 9C, FIG. 10, FIG. 11, FIG. 12, Table 5, and any othervector encompassed by the present application. In particularembodiments, a derivative vector (1) may be obtained by alteration of avector represented in FIG. 1A, FIG. 1B, FIG. 2A, FIG. 6A, FIG. 6B, FIG.8A, FIG. 8B, FIG. 8C, FIG. 8D, FIG. 9A, FIG. 9B, FIG. 9C, FIG. 10, FIG.11, FIG. 12, Table 5, and any other vector encompassed by the presentapplication, or (2) may contain one or more elements (e.g., antibioticresistance marker, recombination or restriction site, etc.) of a vectorrepresented in FIG. 1A, FIG. 1B, FIG. 2A, FIG. 6A, FIG. 6B, FIG. 8A,FIG. 8B, FIG. 8C, FIG. 8D, FIG. 9A, FIG. 9B, FIG. 9C, FIG. 10, FIG. 11,FIG. 12, Table 5, and any other vector encompassed by the presentapplication. Further, as noted above, a derivative vector may containone or more element which shares sequence similarity (e.g., at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, etc. sequence identity at the nucleotide level) to one or moreelement of a vector represented in FIG. 1A, FIG. 1B, FIG. 2A, FIG. 6A,FIG. 6B, FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D, FIG. 9A, FIG. 9B, FIG. 9C,FIG. 10, FIG. 11, FIG. 12, Table 5, and any other vector encompassed bythe present application. Derivative vectors may also share at least atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, etc. sequence identity at the nucleotide level to thecomplete nucleotide sequence of a vector represented in FIG. 1A, FIG.1B, FIG. 2A, FIG. 6A, FIG. 6B, FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D, FIG.9A, FIG. 9B, FIG. 9C, FIG. 10, FIG. 11, FIG. 12, Table 5, and any othervector encompassed by the present application. Derivative vectorsinclude those which have been generated by performing a cloning reactionupon a vector represented in FIG. 1A, FIG. 1B, FIG. 2A, FIG. 6A, FIG.6B, FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D, FIG. 9A, FIG. 9B, FIG. 9C, FIG.10, FIG. 11, FIG. 12, Table 5, and any other vector encompassed by thepresent application. Derivative vectors also include vectors which havebeen generated by the insertion into another vector of one or morestructural and/or functional components of a vector (e.g. one or moregenes or portions thereof encoding one or more structural or functionalproteins (or portions thereof) of a vector), including but not limitedto the vectors represented in FIG. 1A, FIG. 1B, FIG. 2A, FIG. 6A, FIG.6B, FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D, FIG. 9A, FIG. 9B, FIG. 9C, FIG.10, FIG. 11, FIG. 12, Table 5, and any other vector encompassed by orsuitable for use in the invention. Often these derivative vectors willcontain at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, etc. of the nucleic acid present in a vectorrepresented in FIG. 1A, FIG. 1B, FIG. 2A, FIG. 6A, FIG. 6B, FIG. 8A,FIG. 8B, FIG. 8C, FIG. 8D, FIG. 9A, FIG. 9B, FIG. 9C, FIG. 10, FIG. 11,FIG. 12, Table 5, and any other vector encompassed by the presentapplication. Derivative vectors also include progeny of any of thevectors referred to above, as well as vectors referred to above whichhave been subjected to mutagenesis (e.g., random mutagenesis).

Promoter: As used herein, a promoter is an example of a transcriptionalregulatory sequence, and is specifically a nucleic acid sequencegenerally described as the proximal region of a gene located 5′ to thestart codon. The transcription of an adjacent nucleic acid segment isinitiated at the promoter region. A repressible promoter's rate oftranscription decreases in response to a repressing agent. An induciblepromoter's rate of transcription increases in response to an inducingagent. A constitutive promoter's rate of transcription is notspecifically regulated, though it can vary under the influence ofgeneral metabolic conditions. Suitable examples of promoters that may beused in the present invention include, but are not limited to polymeraseIII promoters such as H1 and U6.

Product: As used herein, a “product” is one of the desired daughtermolecules produced after cloning process. The product contains thenucleic acid which was to be cloned or subcloned.

Recognition sequence: As used herein, a “recognition sequence”(alternatively and equivalently referred to herein as a recognitionsite) is a particular sequence to which a protein, chemical compound,DNA, or RNA molecule (e.g., restriction endonuclease, a topoisomerase, amodification methylase, a type IIs restriction enzyme, or a recombinase)recognizes and binds. In the present invention, a recognition sequencemay refer to a recombination site (which may alternatively be referredto as a recombinase recognition site), a topoisomerase recognition site,or a type IIs restriction enzyme recognition site. For example, therecognition sequence for Cre recombinase is loxP which is a 34 base pairsequence comprised of two 13 base pair inverted repeats (serving as therecombinase binding sites) flanking an 8 base pair core sequence. SeeFIG. 1 of Sauer, B., Current Opinion in Biotechnology 5:521-527 (1994).Other examples of such recognition sequences are the attB, attP, attL,and attR sequences which are recognized by the recombinase enzyme.Integrase attB is an approximately 25 base pair sequence containing two9 base pair core-type Int binding sites and a 7 base pair overlapregion. attP is an approximately 240 base pair sequence containingcore-type Int binding sites and arm-type Int binding sites as well assites for auxiliary proteins integration host factor (IHF), FIS andexcisionase (Xis). See Landy, Current Opinion in Biotechnology 3:699-707(1993). Such sites may also be engineered according to the presentinvention to enhance production of products in the methods of theinvention. When such engineered sites lack the P1 or H1 domains to makethe recombination reactions irreversible (e.g., attR or attP), suchsites may be designated attR′ or attP′ to show that the domains of thesesites have been modified in some way. Examples of topoisomeraserecognitions sites include, but are not limited to, the sequence5′-GCAACTT-3′ that is recognized by E. coli topoisomerase III (a type Itopoisomerase); the sequence 5′-(C/T)CCTT-3′ which is a topoisomeraserecognition site that is bound specifically by most poxvirustopoisomerases, including vaccinia virus DNA topoisomerase I; and othersthat are known in the art as discussed elsewhere herein.

Recombination proteins: As used herein, “recombination proteins” includeexcisive or integrative proteins, enzymes, co-factors or associatedproteins that are involved in recombination reactions involving one ormore recombination sites, which may be wild-type proteins (See Landy,Current Opinion in Biotechnology 3:699-707 (1993)), or mutants,derivatives (e.g., fusion proteins containing the recombination proteinsequences or fragments thereof), fragments, and variants thereof.Suitable recombination proteins for use in the present inventioninclude, but are not limited to Int, Cre, IHF, Xis, Fis, Hin, Gin, Cin,Tn3 resolvase, TndX, XerC and XerD.

Recombination site: As used herein, a “recombination site” is arecognition sequence on a nucleic acid molecule participating in anintegration/recombination reaction by recombination proteins.Recombination sites are discrete sections or segments of nucleic acid onthe participating nucleic acid molecules that are recognized and boundby a site-specific recombination protein during the initial stages ofintegration or recombination. For example, the recombination site forCre recombinase is loxP which is a 34 base pair sequence comprised oftwo 13 base pair inverted repeats (serving as the recombinase bindingsites) flanking an 8 base pair core sequence. See FIG. 1 of Sauer, B.,Curr. Opin. Biotech. 5:521-527 (1994). Other examples of recognitionsequences include the attB, attP, attL, and attR sequences describedherein, and mutants, fragments, variants and derivatives thereof, whichare recognized by the recombination protein Int and by the auxiliaryproteins integration host factor (IHF), FIS and excisionase (Xis). SeeLandy, Curr. Opin. Biotech. 3:699-707 (1993).

Recombinational Cloning: As used herein, “recombinational cloning” is amethod, such as that described in U.S. Pat. Nos. 5,888,732, 6,143,557,6,171,861, 6,270,969, and 6,277,608 (the contents of which are fullyincorporated herein by reference), whereby segments of nucleic acidmolecules or populations of such molecules are exchanged, inserted,replaced, substituted or modified, in vitro or in vivo. Suitably, suchcloning method is an in vitro method, i.e., a method in which therecombination reaction takes place outside of or in the absence of hostcells.

Selectable marker: As used herein, “selectable marker” is a nucleic acidsegment that allows one to select for or against a molecule (e.g., areplicon) or a cell that contains it, often under particular conditions.These markers can encode an activity, such as, but not limited to,production of RNA, peptide, or protein, or can provide a binding sitefor RNA, peptides, proteins, inorganic and organic compounds orcompositions and the like. Examples of selectable markers include butare not limited to: (1) nucleic acid segments that encode products whichprovide resistance against otherwise toxic compounds (e.g.,antibiotics); (2) nucleic acid segments that encode products which areotherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophicmarkers); (3) nucleic acid segments that encode products which suppressthe activity of a gene product; (4) nucleic acid segments that encodeproducts which can be readily identified (e.g., phenotypic markers suchas β-galactosidase, green fluorescent protein (GFP), yellow fluorescentprotein (YFP), cyan fluorescent protein (CFP), and cell surfaceproteins); (5) nucleic acid segments that bind products which areotherwise detrimental to cell survival and/or function; (6) nucleic acidsegments that otherwise inhibit the activity of any of the nucleic acidsegments described in Nos. 1-5 above (e.g., antisense oligonucleotides);(7) nucleic acid segments that bind products that modify a substrate(e.g. restriction endonucleases); (8) nucleic acid segments that can beused to isolate or identify a desired molecule (e.g. specific proteinbinding sites); (9) nucleic acid segments that encode a specificnucleotide sequence which can be otherwise non-functional (e.g., for PCRamplification of subpopulations of molecules); (10) nucleic acidsegments, which when absent, directly or indirectly confer resistance orsensitivity to particular compounds; and/or (11) nucleic acid segmentsthat encode products which are toxic in recipient cells.

Examples of toxic gene products are well known in the art, and include,but are not limited to, restriction endonucleases (e.g., DpnI),apoptosis-related genes (e.g. ASK1 or members of the bcl-2/ced-9family), retroviral genes including those of the human immunodeficiencyvirus (HIV), defensins such as NP-1, inverted repeats or pairedpalindromic nucleic acid sequences, bacteriophage lytic genes such asthose from (ΦX174 or bacteriophage T4; antibiotic sensitivity genes suchas rpsL, antimicrobial sensitivity genes such as pheS, plasmid killergenes, eukaryotic transcriptional vector genes that produce a geneproduct toxic to bacteria, such as GATA-1, and genes that kill hosts inthe absence of a suppressing function, e.g., kicB, ccdB, ΦX174 E (Liu,Q. et al., Curr. Biol. 8:1300-1309 (1998), and other genes thatnegatively affect replicon stability and/or replication. A toxic genecan alternatively be selectable in vitro, e.g., a restriction site.

Selection scheme: As used herein, “selection scheme” is any method whichallows selection, enrichment, or identification of a desired product orproduct(s). The selection schemes of one suitable embodiment have atleast two components that are either linked or unlinked duringrecombinational cloning. One component is a Selectable marker. The othercomponent controls the expression in vitro or in vivo of the Selectablemarker, or survival of the cell (or the nucleic acid molecule, e.g., areplicon) harboring the plasmid carrying the Selectable marker.Generally, this controlling element will be a repressor or inducer ofthe Selectable marker, but other means for controlling expression oractivity of the Selectable marker can be used. Whether a repressor oractivator is used will depend on whether the marker is for a positive ornegative selection, and the exact arrangement of the various nucleicacid segments, as will be readily apparent to those skilled in the art.

Fragments of selectable markers can be arranged relative to therecombination sites or restriction sites such that when the segments arebrought together, they reconstitute a functional Selectable marker. Forexample, the linking event can link a promoter with a structural nucleicacid molecule (e.g., a gene), can link two fragments of a structuralnucleic acid molecule, or can link nucleic acid molecules that encode aheterodimeric gene product needed for survival, or can link portions ofa replicon.

Site-specific recombinase: As used herein, a “site specific recombinase”is a type of recombinase which typically has at least the following fouractivities (or combinations thereof): (1) recognition of one or twospecific nucleic acid sequences; (2) cleavage of said sequence orsequences; (3) topoisomerase activity involved in strand exchange; and(4) ligase activity to reseal the cleaved strands of nucleic acid. SeeSauer, B., Current Opinions in Biotechnology 5:521-527 (1994).Conservative site-specific recombination is distinguished fromhomologous recombination and transposition by a high degree ofspecificity for both partners. The strand exchange mechanism involvesthe cleavage and rejoining of specific nucleic acid sequences in theabsence of DNA synthesis (Landy, A. (1989) Ann. Rev. Biochem.58:913-949).

Vector: As used herein, a “vector” is a nucleic acid molecule(preferably DNA) that provides a useful biological or biochemicalproperty to an Insert. Examples include plasmids, phages, autonomouslyreplicating sequences (ARS), centromeres, and other sequences which areable to replicate or be replicated in vitro or in a host cell, or toconvey a desired nucleic acid segment to a desired location within ahost cell. A Vector can have one or more restriction endonucleaserecognition sites (whether type I, II or IIs) at which the sequences canbe cut in a determinable fashion without loss of an essential biologicalfunction of the vector, and into which a nucleic acid fragment can bespliced in order to bring about its replication and cloning. Vectors canalso comprise one or more recombination sites that permit exchange ofnucleic acid sequences between two nucleic acid molecules. Such as, forexample, subcloning of genes of interest between Entry and Destinationvectors in the Gateway™ system (available from Invitrogen Corporation,Carlsbad, Calif. (see, e.g., FIG. 36)). Vectors can further provideprimer sites, e.g., for PCR, transcriptional and/or translationalinitiation and/or regulation sites, recombinational signals, replicons,Selectable markers, etc. Clearly, methods of inserting a desired nucleicacid fragment which do not require the use of recombination,transpositions or restriction enzymes (such as, but not limited to, UDGcloning of PCR fragments (U.S. Pat. No. 5,334,575, entirely incorporatedherein by reference), TA Cloning® brand PCR cloning (InvitrogenCorporation, Carlsbad, Calif.) (also known as direct ligation cloning),and the like) can also be applied to clone a fragment into a cloningvector to be used according to the present invention. The cloning vectorcan further contain one or more selectable markers suitable for use inthe identification of cells transformed with the cloning vector.

Incorporating: As used herein, “incorporating” means becoming a part ofa nucleic acid (e.g., DNA) molecule or primer.

Nucleotide: As used herein, a “nucleotide” is a base-sugar-phosphatecombination. Nucleotides are monomeric units of a nucleic acid molecule(DNA and RNA). The term nucleotide includes ribonucleoside triphosphatesATP, UTP, CTG, GTP and deoxyribonucleoside triphosphates such as dATP,dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. The termnucleotide as used herein also refers to dideoxyribonucleosidetriphosphates (ddNTPs) and their derivatives. Illustrated examples ofdideoxyribonucleoside triphosphates include, but are not limited to,ddATP, ddCTP, ddGTP, ddITP, and ddTTP. According to the presentinvention, a “nucleotide” may be unlabeled or detectably labeled by wellknown techniques. Detectable labels include, for example, radioactiveisotopes, fluorescent labels, chemiluminescent labels, bioluminescentlabels and enzyme labels.

Portion: As used herein, the term “portion” refers to part, orpercentage of a whole entity. For example, a “portion” of a nucleic acidmolecule refers to 1%, 10%, 25%, 50%, 75%, 90%, 99%, etc., of the wholenucleic acid molecule.

Segment: As used herein, the term “segment” refers to part, orpercentage of a whole entity. For example, a “segment” of a nucleic acidmolecule refers to 1%, 10%, 25%, 50%, 75%, 90%, 99%, etc., of the wholenucleic acid molecule.

Other terms used in the fields of recombinant nucleic acid technologyand molecular and cell biology as used herein will be generallyunderstood by one of ordinary skill in the applicable arts.

The present invention relates to methods, compositions, isolated nucleicacids, vectors and kits for seamless cloning of nucleic acid moleculesand production of nucleic acids and proteins.

The vectors represented througout, specifically shown in FIGS. 1A, 1B,2A, 6A and 6B, 8A, 8B, 8C, 8D, 9A, 9B, 9C, 10, 11, 28, 33, 37 as well assimilar vectors and portions of these vectors, may be used in thepractice of the methods of the present invention. In each case, thesevectors are designed such that upon digestion with a restriction enzyme(e.g. a type IIs restriction enzyme), a sticky end is generated abuttingand/or including nucleic acids which encode a peptide which may becleaved from a protein or peptide encoded by a nucleic acid which isinserted into the vector. These, and other vectors of the presentinvention may further comprise one or more signal peptides and/orprotease cleavage sites. The vectors of the present invention allow forthe production of a protein that is exported from a cell and cleaved togenerate a “mature” protein. The vectors of the present invention alsoallow for the production of a protein that is retained in the cell as a“native” protein.

In one aspect, the present invention provides methods for joining one ormore (e.g. one, two, three, four, five, etc.) first nucleic acidmolecules and a second one or more nucleic acid molecules, comprising:(a) combining the first and second nucleic acid molecules underconditions sufficient to allow for the joining of at least one terminusof the first nucleic acid molecule(s) to at least one terminus of thesecond nucleic acid molecule(s), wherein the terminus of the firstnucleic acid molecule which is connected to the terminus of the secondnucleic acid molecule(s) comprises a sticky end (e.g. an overhangingend) generated by a restriction enzyme (e.g. a type IIs restrictionenzyme) and the terminus of the second nucleic acid molecule(s) iscompatible (e.g. a blunt end or a sticky end) with this sticky end. Inembodiments similar to the above and elsewhere herein, the sticky end mybe on the terminus of the second nucleic acid molecule and the firstnucleic acid molecule may contain a compatible end.

As in other embodiments of the invention described herein, the secondnucleic acid molecule may contain an end which is generated by digestionwith a type IIs restriction enzyme and the first nucleic acid moleculemay contain a compatible end generated by other means.

In suitable embodiments, the present invention provides methods ofcloning or subcloning one or more desired nucleic acid moleculescomprising: (a) combining in vitro or in vivo, (i) one or more firstnucleic acid molecules comprising one or more sticky ends that have beengenerated by one or more restriction enzymes (e.g. one or more type IIsrestriction enzymes); and (ii) one or more second nucleic acid moleculescomprising one or more ends which are compatible with the one or moresticky ends on the first nucleic acid molecule(s) and, optionally, oneor more selectable markers; and (b) incubating the combination underconditions sufficient to join the first nucleic acid molecule and one ormore of the second nucleic acid molecules, thereby producing one or moredesired product nucleic acid molecules.

In another aspect, the present invention provides methods for cloning orsubcloning one or more desired nucleic acid molecules comprising: (a)combining in vitro or in vivo, (i) one or more first nucleic acidmolecules comprising one or more sticky ends that have been generated byone or more restriction enzymes (e.g. one or more type IIs restrictionenzymes); (ii) one or more second nucleic acid molecules comprising oneor more restriction sites (e.g. one or more first type IIs restrictionenzyme recognition sites) and, optionally, one or more selectablemarkers; and (iii) one or more restriction enzymes (e.g., one or moretype IIs restriction enzymes) that are specific for the restrictionenzyme recognition site; and (b) incubating the combination underconditions sufficient to join the first nucleic acid molecule and one ormore of the second nucleic acid molecules, thereby producing one or moredesired product nucleic acid molecules.

In another aspect, the present invention provides methods for cloning orsubcloning one or more desired nucleic acid molecules, or portionsthereof, comprising: (a) combining in vitro or in vivo, (i) one or morefirst nucleic acid molecules comprising at least one nucleic acidsegment that is flanked by one or more restriction sites (e.g. one ormore first type IIs restriction enzyme recognition sites); (ii) one ormore second nucleic acid molecules comprising one or more ends which arecompatible with a sticky end on the segment and, optionally, one or moreselectable markers; and (iii) one or more restriction enzymes (e.g., oneor more type IIs restriction enzymes) that are specific for therestriction enzyme recognition site; and (b) incubating the combinationunder conditions sufficient to join the first nucleic acid segment andone or more of the second nucleic acid molecules, thereby producing oneor more desired product nucleic acid molecules.

In another aspect, the present invention provides methods for cloning orsubcloning one or more desired nucleic acid molecules, or portionsthereof, comprising: (a) combining in vitro or in vivo, (i) one or morefirst nucleic acid molecules comprising at least one nucleic acidsegment that is flanked by one or more first restriction sites (e.g. oneor more first type IIs restriction enzyme recognition sites); (ii) oneor more second nucleic acid molecules comprising one or more secondrestriction sites (e.g. one or more type IIs restriction enzymerecognition sites) and, optionally, one or more selectable markers; and(iii) one or more restriction enzymes (e.g. one or more type IIsrestriction enzymes) that are specific for the first and/or second typeIIs restriction enzyme recognition sites; and (b) incubating thecombination under conditions sufficient to join the first nucleic acidsegment and one or more of the second nucleic acid molecules, therebyproducing one or more desired product nucleic acid molecules.

The seamless cloning methods of the present invention may utilize anyrestriction enzyme, including those which cleave nucleic acid moleculesto produce blunt ends. The term “blunt ends” as used herein is used toindicate a nucleic acid molecule which has been cleaved by a restrictionenzyme in such a way as to produce a double stranded nucleic acid inwhich both strands stop “bluntly” and do not overlap or overhang theother. Suitably, the methods of the invention utilize type IIsrestriction sites. The present invention also encompasses the use ofblunt-end cleavage enzymes, such as, but not limited to, ScaI, SmaI,HpaI, HincII, HaeII and AluI.

Type-IIs restriction enzymes and recognition sites which are useful inall aspects of the present invention include, but are not limited to,EarI, MnlI, PleI, AlwI, BbsI, BsaI, BsmAI, BspMI, Esp3I, HgaI, SapI,SfaNI, BbvI, BsmFI, FokI, BseRI, HphI, Alw26I, BbvlI, BpmI, BsmI, BbsI,BsmBI, BaeI, BsrI, MlyI, BsrDI, Eco57I, GsuI, Mn1I, PleI, TaqII,Tth111II and MboII. In all aspects of the present invention, therestriction enzyme recognition sites on the first and second nucleicacid molecules may be the same sites or they may be different. Inaddition, the restriction enzyme recognition sites may be the same ordifferent on each nucleic acid molecule. This allows for selectivecloning where only nucleic acid segments with complementary sites willtransfer between nucleic acids molecules.

Cleavage of a polynucleotide sequence with a type IIs restriction enzymeleaves an overhang on one strand of the sequence, or a sticky end. Viathe cloning methods of the present invention, this sticky end can becombined with a compatible sequence on a second nucleic acid moleculeresulting in a cloned, co-joined molecule. Sequences cleaved by Type IIssites may also be joined to blunt ended compatible nucleic acidsequences via the cloning methods of the present invention. Thecompatible sequences can be joined via various catalyzing enzymes, forexample DNA ligase and topoisomerase. Certain type IIs enzymes (e.g.MlyI) cleave and leave a blunt end on a nucleic acid molecule that maythen be combined with a sticky end on a second nucleic acid molecule.

Nucleic acid molecules of the invention to be cloned may contain a bluntend to be linked, and the second nucleic acid molecule involved in thecloning method may contain an overhang at the end which is to be linkedby a site-specific topoisomerase (e.g., a type IA or a type IBtopoisomerase), wherein the overhang includes a sequence complementaryto that comprising the blunt end, thereby facilitating strand invasionas a means to properly position the ends for the linking reaction.

The nucleic acid molecules generated using this aspect of the inventioninclude those in which one strand (not both strands) is covalentlylinked at the ends to be linked (i.e. double-stranded nucleic acidmolecules generated using these methods contain a nick at each positionwhere two ends were joined). These embodiments are particularlyadvantageous in that a polymerase can be used to replicate thedouble-stranded (ds) nucleic acid molecule by initially replicating thecovalently linked strand. For example, a thermostable polymerase such asa polymerase useful for performing an amplification reaction such as PCRcan be used to replicate the covalently strand, whereas the strandcontaining the nick does not provide a suitable template forreplication.

Preferably, the 5′ termini of the ends of the nucleotide sequences to belinked by a type IB topoisomerase according to a method of certainaspects of the invention contain complementary 5′ overhanging sequences,which can facilitate the initial association of the nucleotidesequences, including, if desired, in a predetermined directionalorientation. Alternatively, the 5′ termini of the ends of the nucleotidesequences to be linked by a type LB topoisomerase according to a methodof certain aspects of the invention contain complementary 5′ sequenceswherein one of the sequences contains a 5′ overhanging sequence and theother nucleotide sequence contains a complementary sequence at a bluntend of a 5′ terminus, to facilitate the initial association of thenucleotide sequences through strand invasion, including, if desired, ina predetermined directional orientation. The term “5′ overhang” or “5′overhanging sequence” is used herein to refer to a strand of a nucleicacid molecule that extends in a 5′ direction beyond the terminus of thecomplementary strand of the nucleic acid molecule. Conveniently, a 5′overhang can be produced as a result of site specific cleavage of anucleic acid molecule by a type IB topoisomerase.

Preferably, the 3′ termini of the ends of the nucleotide sequences to belinked by a type IA topoisomerase according to a method of certainaspects of the invention contain complementary 3′ overhanging sequences,which can facilitate the initial association of the nucleotidesequences, including, if desired, in a predetermined directionalorientation. Alternatively, the 3′ termini of the ends of the nucleotidesequences to be linked by a topoisomerase (e.g., a type IA or a type IItopoisomerase) according to a method of certain aspects of the inventioncontain complementary 3′ sequences wherein one of the sequences containsa 3′ overhanging sequence and the other nucleotide sequence contains acomplementary sequence at a blunt end of a 3′ terminus, to facilitatethe initial association of the nucleotide sequences through strandinvasion, including, if desired, in a predetermined directionalorientation. The term “3 overhang” or “3 overhanging sequence” is usedherein to refer to a strand of a nucleic acid molecule that extends in a5′ direction beyond the terminus of the complementary strand of thenucleic acid molecule. Conveniently, a 3′ overhang can be produced uponcleavage by a type IA or type II topoisomerase.

The cloning methods of the present invention may be performed in vitroor in vivo. By in vitro and in vivo herein is meant cloning that iscarried out outside of host cells (e.g., in cell-free systems, or insystems containing host cells in which the various cloning andrecombination reaction(s) of the present invention take(s) place outsideof the host cells) or inside of host cells (e.g., using recombination orother proteins expressed by host cells), respectively.

The nucleic acid molecules utilized and produced in the methods,compositions and kits of the present invention may be vectors or linearnucleic acid molecules. The term “vector,” as used herein, refers to anucleic acid molecule (preferably DNA) that provides a useful biologicalor biochemical property to an inserted nucleic acid. The terms “vector”and “plasmid” are used interchangeably herein. Examples of vectorsinclude, phages, autonomously replicating sequences (ARS), centromeres,and other sequences which are able to replicate or be replicated invitro or in a cell, or to convey a desired nucleic acid segment to adesired location within a cell of an animal. Vectors useful in thepresent invention include chromosomal-, episomal- and virus-derivedvectors, e.g., vectors derived from bacterial plasmids orbacteriophages, and vectors derived from combinations thereof, such ascosmids and phagemids. A vector can have one or more restrictionendonuclease recognition sites at which the sequences can be cut in adeterminable fashion without loss of an essential biological function ofthe vector, and into which a nucleic acid fragment can be spliced inorder to bring about its replication and cloning. Vectors can furtherprovide primer sites, e.g., for PCR, transcriptional and/ortranslational initiation and/or regulation sites, recombinationalsignals, replicons, selectable markers, etc. Clearly, methods ofinserting a desired nucleic acid fragment which do not require the useof homologous recombination, transpositions or restriction enzymes (suchas, but not limited to, UDG cloning of PCR fragments (U.S. Pat. No.5,334,575, entirely incorporated herein by reference), TA Cloning® brandPCR cloning (Invitrogen Corp., Carlsbad, Calif.), and the like) can alsobe applied to clone a nucleic acid into a vector to be used according tothe present invention. The vector can optionally further contain one ormore selectable markers suitable for use in the identification of cellstransformed with the vector, such as the selectable markers and reportergenes described herein. Vectors of the present invention may bederivative vectors as described throughout the present specification.

Vectors known in the art and those commercially available (and variantsor derivatives thereof) may be used in the present invention. Suchvectors may be obtained from, for example, Vector Laboratories Inc.,Invitrogen, Promega, Novagen, NEB, Clontech, Boehringer Mannheim,Pharmacia, EpiCenter, OriGenes Technologies Inc., Stratagene,PerkinElmer, Pharmingen, and Research Genetics. General classes ofvectors of particular interest include prokaryotic and/or eukaryoticcloning vectors, expression vectors, fusion vectors, two-hybrid orreverse two-hybrid vectors, shuttle vectors for use in different hosts,mutagenesis vectors, transcription vectors, vectors for receiving largeinserts and the like.

Other vectors of interest include viral origin vectors (M13 vectors,bacterial phage λ vectors, adenovirus vectors, and retrovirus vectors),high, low and adjustable copy number vectors, vectors which havecompatible replicons for use in combination in a single host (pACYC184and pBR322) and eukaryotic episomal replication vectors (pCDM8).

Vectors for use in the present invention may comprise all, or portionsof viral genomes, for example an adenovirus genome, a baculovirusgenome, a herpesvirus genome, a pox virus genome, an adeno-associatedvirus genome, a retrovirus genome, a flavivirus genome, a togavirusgenome, an alphavirus genome, an RNA virus genome, etc.

The present invention also encompasses the use of recombinantretroviruses, e.g., lentiviruses, or any other type of retrovirus may beused in an analogous fashion to practice the present invention. Acommercially available system for the construction of recombinantlentiviruses is ViraPower™ Lentiviral Expression System, available fromInvitrogen Corporation, Carlsbad, Calif. The ViraPower™ system providesa retroviral system for high-level expression in dividing andnon-dividing eukaryotic cells, e.g., mammalian cells (See FIG. 29).Examples of products available from Invitrogen Corporation, Carlsbad,Calif. include the ViraPower™ Lentiviral Directional TOPO® ExpressionKit (catalog number K4950-00), the ViraPower™ Lentiviral GATEWAY™Expression Kit (catalog number K4960-00), and the ViraPower™ LentiviralSupport Kit (catalog number K4970-00).

The present invention also encompasses replication-incompetentlentiviruses that can deliver and express one or more sequences ofinterest (e.g., genes). These viruses (based loosely on HIV-1) caneffectively transduce dividing and non-dividing mammalian cells (inculture or in vivo), thus broadening the possible applications beyondthose of traditional Moloney (MLV)-based retroviral systems (Clontech,Stratagene, etc.). Directional TOPO and GATEWAY™ lentiviral vectors havebeen created to clone one or more genes of interest with a V5 epitope,if desired. The Directional TOPO method involves a 5 minute bench-topligation and results in 95% correct orientation (See FIGS. 33 and 34).The GATEWAY™ method involves cloning and sequencing a gene of interestonly once into an entry clone and rapidly shuttling the gene of interestfrom vector to vector, or the destination clones. The GATEWAY™ methodrequires no restriction digests, gel purification or ligase. TheGATEWAY™ method is 90-100% efficient and accurate and the gene ofinterest is cloned in the right direction and in-frame (FIG. 35). Thevectors also carry the blasticidin resistance gene (bsd) to allow forthe selection of transduced cells. Without additional modifications,these vectors can theoretically accommodate up to ˜6 kb of foreign gene.Three supercoiled packaging plasmids (gag/pol, rev and VSV-G envelope)are provided to supply helper functions and viral proteins in trans (SeeFIGS. 30 and 32). Finally, an optimized producer cell line (293FT) isprovided that will facilitate production of high titer virus. AnOverview of lentiviral production is summarized in FIG. 31 and involvesthe following steps: 1) Co-transfect 3 packaging plasmids andpLenti6-GOI into 293FT; 2) VSV-G envelope becomes studded in cellmembrane; 3) Rev transports viral genome RNA with gene of interest outof the nucleus; 4) gag protein packages: viral RNA and pol protein; 5)Virus buds off cell, picks up envelope (pseudotyping). Plasmid maps ofvectors adapted for use with GATEWAY™ and topoisomerase cloning in theproduction of nucleic acid molecules comprising all or a portion of alentiviral genome are shown in FIGS. 8A (pLenti6/V5-DEST), 8B(pLenti6/V5-D-TOPO®), 8C (pLenti4/V5-DEST), and 8D (pLenti6/UbC/V5-DEST)respectively. The nucleotide sequences of the plasmids are provided inTables 6-9, SEQ ID NOS:2-5. Plasmid maps of the three packaging plasmidspLP1, pLP2, and pLP/VSVG are shown in FIGS. 9A, 9B, and 9C respectivelyand the nucleotide sequences of these plasmids are provided as Tables10, 11 and 12, (SEQ ID NOS:6-8) respectively.

Retroviruses are RNA viruses that reverse transcribe their genome andintegrate the DNA copy into a chromosome of the target cell. It wasdiscovered that the retroviral packaging proteins (gag, pol and env)could be supplied in trans, thus allowing the creation of replicationincompetent viral particles capable of stably delivering a gene ofinterest. These retroviral vectors have been available for gene deliveryfor many years (Miller et al., (1989) BioTechniques 7:980-990). Onesignificant advantage of retroviral-based delivery is that the gene ofinterest is stably integrated into the genome of the host cell with veryhigh efficiency. In addition, no viral genes are expressed in theserecombinant vectors making them safe to use both in vitro and in vivo.However, one main drawback to the traditional Moloney-based retrovirusesis that the target cell must undergo one round of cell division fornuclear import and stable integration to occur. Traditional retrovirusesdo not have an active mechanism of nuclear import and therefore mustwait for the host cell nuclear membrane to breakdown during mitosisbefore they can access the host genomic DNA (Miller et al., Mol. Cell.Biol. 10:4239-442 (1990)).

Unlike traditional retroviruses, HIV (classified as a “lentivirus”) isactively imported into the nuclei of non-dividing cells (Lewis et al.,J. Virol. 68:510-516 (1994)). HIV still goes through the basicretrovirus lifecycle (RNA genome reverse transcribed in the target celland integrated into the host genome); however, cis-acting elementsfacilitate active nuclear import, allowing HIV to stably infectnon-dividing cells (for reviews see Buchschacher et al., Blood95:2499-2504 (2000), Naldini et al., “The Development of Human GeneTherapy”, Cold Spring Harbor Laboratory Press, pages 47-60 (1999)). Itis important to note that, for both lentivirus and traditionalretroviruses, no gene expression occurs until after the viral RNA genomehas been reverse transcribed and integrated into the host genome.

Similar to other retrovirus expression systems, the packaging functionsof HIV can be supplied in trans, allowing the creation of lentiviralvectors for gene delivery. With all the viral proteins removed, the genedelivery vector becomes safe to use and allows foreign DNA to beefficiently packaged. In addition, it has been shown that lentiviral (orany retroviral) envelope proteins can be substituted for ones withbroader tropism. The substitution of envelope is called pseudotyping,and allows creation of lentiviral vectors capable of infecting a widervariety of cells besides just CD4+ cells. Many have found that the Gprotein from vesicular stomatitis virus (VSV-G) is an excellentpseudotyping envelope protein that imparts a very broad host range forthe virus (Yee et al., Proc. Natl. Acad. Sci. USA 91:9564-9568 (1994)).The ability of pseudo-typed lentivirus to infect a broad range ofnon-dividing cells has led to its extensive use in animal gene deliveryand gene therapy (Baek et al., Hum. Gene Ther. 12:1551-8 (2001), Park etal., Mol. Ther. 4:164-73 (2001), Peng et al., Gene Ther. 8:1456-63(2001)).

The present invention also encompasses the use of adenoviral vectors,including but not limited to, a pAd/PL-DEST vector (Table 11, FIG. 10,SEQ ID NO:7) and pAd/CMV/V5-DEST vector (Table 12, FIG. 11, SEQ IDNO:8). Adenoviruses are non-enveloped viruses with a 36 kb DNA genomethat encodes more than 30 proteins. At the ends of the genome areinverted terminal repeats (ITRs) of approximately 100-150 base pairs. Asequence of approximately 300 base pairs located next to the 5′-ITR isrequired for packaging of the genome into the viral capsid. The genomeas packaged in the virion has terminal proteins covalently attached tothe ends of the linear genome.

The genes encoded by the adenoviral genome are divided into early andlate genes depending upon the timing of their expression relative to thereplication of the viral DNA. The early genes are expressed from fourregions of the adenoviral genome termed E1-E4 and are transcribed priorto onset of DNA replication. Multiple genes are transcribed from eachregion. Portions of the adenoviral genome may be deleted withoutaffecting the infectivity of the deleted virus. The genes transcribedfrom regions E1, E2, and E4 are essential for viral replication whilethose from the E3 region may be deleted without affecting replication.The genes from the essential regions can be supplied in trans to allowthe propagation of a defective virus. For example, deletion of the E1region of the adenoviral genome results in a virus that is replicationdefective. Viruses deleted in this region are grown on 293 cells thatexpress the viral E1 genes from the genome of the cell.

In addition to permitting the construction of a safer,replication-defective viruses, deletion and complementation in trans ofportions of the adenoviral genome and/or deletion of non-essentialregions make space in the adenoviral genome for the insertion ofheterologous DNA sequences. The packaging of viral DNA into a viralparticle is size restricted with an upper limit of approximately 38 kbof DNA. In order to maximize the amount of heterologous DNA that may beinserted and packaged, viruses have been constructed that lack all ofthe viral genome except the ITRs and packaging sequence (see, U.S. Pat.No. 6,228,646). All of the viral functions necessary for replication andpackaging are provided in trans from a defective helper virus that isdeleted in the packaging signal.

The present invention also encompasses the use of herpes viruses (see,for example, U.S. Pat. No. 5,672,344, issued to Kelly, et al.). Thefamily Herpesviridae contains three subfamilies 1) alphaherpesvirinae,containing among others human herpesvirus 1; 2) betaherpesvirinae,containing the cytomegaloviruses; and 3) gammaherpesvirinae.Herpesviruses are enveloped DNA viruses. Herpesviruses form particlesthat are approximately spherical in shape and that contain one moleculeof linear dsDNA and approximately 20 structural proteins. Numerousherpesviruses have been isolated from a wide variety of hosts. Forexample, U.S. Pat. No. 6,121,043 issued to Cochran, et al. describesrecombinant herpesvirus of turkeys comprising a foreign DNA insertedinto a non-essential region of the herpesvirus of turkeys genome; U.S.Pat. No. 6,410,311 issued to Cochran, et al. describes recombinantfeline herpesvirus comprising a foreign DNA inserted into a regioncorresponding to a 3.0 kb EcoRI-SalI fragment of a feline herpesvirusgenome, U.S. Pat. No. 6,379,967 issued to Meredith, et al., describesherpesvirus saimiri, (HVS; a lymphotropic virus of squirrel monkeys) asa viral vector; and U.S. Pat. No. 6,086,902 issued to Zamb, et al.describes recombinant bovine herpesvirus type 1 vaccines.

Herpesviruses have been used as vectors to deliver exogenous nucleicacid material to a host cell. In addition to the examples above, U.S.Pat. No. 4,859,587, issued to Roizman describes recombinant herpessimplex viruses, vaccines and methods, U.S. Pat. No. 5,998,208 issued toFraefel, et al., describes a helper virus-free herpesvirus vectorpackaging system, U.S. Pat. No. 6,342,229 issued to O'Hare, et al.,describes herpesvirus particles comprising fusion protein and theirpreparation and use and U.S. Pat. No. 6,319,703 issued to Speckdescribes recombinant virus vectors that include a double mutantherpesvirus such as an herpes simplex virus-1 (HSV-1) mutant lacking theessential glycoprotein gH gene and having a mutation impairing thefunction of the gene product VP16.

Suitable vectors for use in the present invention also includeprokaryotic vectors such as pcDNA II, pSL301, pSE280, pSE380, pSE420,pTrcHisA, B, and C, pRSET A, B, and C (Invitrogen, Corp.), pGEMEX-1, andpGEMEX-2 (Promega, Inc.), the pET vectors (Novagen, Inc.), pTrc99A,pKK223-3, the pGEX vectors, pEZZ18, pRIT2T, and pMC1871 (Pharmacia,Inc.), pKK233-2 and pKK388-1 (Clontech, Inc.), and pProEx-HT(Invitrogen, Corp.) and variants and derivatives thereof. Other vectorsof interest include eukaryotic expression vectors such as pFastBac,pFastBacHT, pFastBacDUAL, pSFV, and pTet-Splice (Invitrogen), pEUK-C1,pPUR, pMAM, pMAMneo, pBI101, pBI121, pDR2, pCMVEBNA, and pYACneo(Clontech), pSVK3, pSVL, pMSG, pCH110, and pKK232-8 (Pharmacia, Inc.),p3′SS, pXT1, pSG5, pPbac, pMbac, pMC1neo, and pOG44 (Stratagene, Inc.),and pYES2, pAC360, pBlueBacHis A, B, and C, pVL1392, pBlueBacIII, pCDM8,pcDNA1, pZeoSV, pcDNA3 pREP4, pCEP4, and pEBVHis (Invitrogen, Corp.) andvariants or derivatives thereof.

Other vectors suitable for use in the invention include pUC18, pUC19,pBlueScript, pSPORT, cosmids, phagemids, YAC's (yeast artificialchromosomes), BAC's (bacterial artificial chromosomes), P1 (Escherichiacoli phage), pQE70, pQE60, pQE9 (quagan), pBS vectors, PhageScriptvectors, BlueScript vectors, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene),pcDNA3 (Invitrogen), pGEX, pTrsfus, pTrc99A, pET-5, pET-9, pKK223-3,pKK233-3, pDR540, pRIT5 (Pharmacia), pSPORT1, pSPORT2, pCMVSPORT2.0 andpSV-SPORT1 (Invitrogen) and variants or derivatives thereof.

Additional vectors of interest include pTrxFus, pThioHis, pLEX, pTrcHis,pTrcHis2, pRSET, pBlueBacHis2, pcDNA3.1/His, pcDNA3.1(−)/Myc-His,pSecTag, pEBVHis, pPIC9K, pPIC3.5K, pAO815, pPICZ, pPICZα, pGAPZ,pGAPZα, pBlueBac4.5, pBlueBacHis2, pMelBac, pSinRep5, pSinHis, pIND,pIND(SP1), pVgRXR, pcDNA2.1, pYES2, pZErO1.1, pZErO-2.1, pCR-Blunt,pSE280, pSE380, pSE420, pVL1392, pVL1393, pCDM8, pcDNA1.1, pcDNA1.1/Amp,pcDNA3.1, pcDNA3.1/Zeo, pSe, SV2, pRc/CMV2, pRc/RSV, pREP4, pREP7,pREP8, pREP9, pREP 10, pCEP4, pEBVHis, pCR3.1, pCR2.1, pCR3.1-Uni, andpCRBac from Invitrogen; λ ExCell, λ gt11, pTrc99A, pKK223-3, pGEX-1λT,pGEX-2T, pGEX-2TK, pGEX-4T-1, pGEX-4T-2, pGEX-4T-3, pGEX-3X, pGEX-5X-1,pGEX-5X-2, pGEX-5X-3, pEZZ18, pRIT2T, pMC1871, pSVK3, pSVL, pMSG,pCH110, pKK232-8, pSL1180, pNEO, and pUC4K from Pharmacia;pSCREEN-1b(+), pT7Blue(R), pT7Blue-2, pCITE-4-abc(+), pOCUS-2, pTAg,pET-32LIC, pET-30LIC, pBAC-2 cp LIC, pBACgus-2cp LIC, pT7Blue-2 LIC,pT7Blue-2, λSCREEN-1, λBlueSTAR, pET-3abcd, pET-7abc, pET9abcd,pET11abcd, pET12abc, pET-14b, pET-15b, pET-16b, pET-17b-pET-17xb,pET-19b, pET-20b(+), pET-21abcd(+), pET-22b(+), pET-23abcd(+),pET-24abcd(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28abc(+),pET-29abc(+), pET-30abc(+), pET-31b(+), pET-32abc(+), pET-33b(+),pBAC-1, pBACgus-1, pBAC4x-1, pBACgus4x-1, pBAC-3cp, pBACgus-2cp,pBACsurf-1, plg, Signal plg, pYX, Selecta Vecta-Neo, Selecta Vecta-Hyg,and Selecta Vecta-Gpt from Novagen; pLexA, pB42AD, pGBT9, pAS2-1,pGAD424, pACT2, pGAD GL, pGAD GH, pGAD10, pGilda, pEZM3, pEGFP, pEGFP-1,pEGFP-N, pEGFP-C, pEBFP, pGFPuv, pGFP, p6xHis-GFP, pSEAP2-Basic,pSEAP2-Contral, pSEAP2-Promoter, pSEAP2-Enhancer, pβgal-Basic,pβgal-Control, pβgal-Promoter, pβgal-Enhancer, pCMVβ, pTet-Off, pTet-On,pTK-Hyg, pRetro-Off, pRetro-On, pIRES1neo, pIRES1hyg, pLXSN, pLNCX,pLAPSN, pMAMneo, pMAMneo-CAT, pMAMneo-LUC, pPUR, pSV2neo, pYEX4T-1/2/3,pYEX-S1, pBacPAK-His, pBacPAK8/9, pAcUW31, BacPAK6, pTrip1Ex, λgt10,λgt11, pWE15, and λTrip1Ex from Clontech; Lambda ZAP II, pBK-CMV,pBK-RSV, pBluescript II KS +/−, pBluescript II SK +/−, pAD-GAL4,pBD-GAL4 Cam, pSurfscript, Lambda FIX II, Lambda DASH, Lambda EMBL3,Lambda EMBL4, SuperCos, pCR-Scrigt Amp, pCR-Script Cam, pCR-ScriptDirect, pBS +/−, pBC KS +/−, pBC SK +/−, Phagescript, pCAL-n-EK, pCAL-n,pCAL-c, pCAL-kc, pET-3abcd, pET-11abcd, pSPUTK, pESP-1, pCMVLacI,pOPRSVI/MCS, pOPI3 CAT, pXT1, pSG5, pPbac, pMbac, pMC1neo, pMC1neo PolyA, pOG44, pOG45, pFRTβGAL, pNEOβGAL, pRS403, pRS404, pRS405, pRS406,pRS413, pRS414, pRS415, and pRS416 from Stratagene.

Two-hybrid and reverse two-hybrid vectors of interest include pPC86,pDBLeu, pDBTrp, pPC97, p2.5, pGAD1-3, pGAD10, pACt, pACT2, pGADGL,pGADGH, pAS2-1, pGAD424, pGBT8, pGBT9, pGAD-GAL4, pLexA, pBD-GAL4,pHISi, pHISi-1, placZi, pB42AD, pDG202, pJK202, pJG4-5, pNLexA, pYESTrpand variants or derivatives thereof.

The present invention also embodies the use and production of chimericvectors. Such chimeric vectors may comprise one or more sequences thatencode one or more functional or structural component of a viral vector,wherein each component may or may not come from the same or differenttypes of viruses. Suitable components that may be combined to createsuch a chimeric vector include, but are not limited to, gag, poi, env,and rev genes and capsid proteins.

The nucleic acid molecules produced and/or utilized in the cloningmethods, compositions and kits of the present invention may additionallyor alternatively comprise one or more promoter molecules as describedthroughout the present specification, including the Pol III promoters H1and U6 as well as other promoters recognized by RNA polymerase III. Thenucleic acid molecules and vectors of the present invention may alsofurther or alternatively comprise one or more genes which code forsignal peptides and/or protease cleavage sites. Examples of proteasecleavage sites include, but are not limited to, TEV sites and EK sites.TEV cleavage sites useful in the present invention include:

(SEQ ID NO: 23) Consensus sequence: Glu-Xaa-Xaa-Try-Xaa-Gln//Xaa¹ (SEQID NO: 24) TEV1: Glu-Asn-Leu-Try-Phe-Gln//Xaa¹ (SEQ ID NO: 25) TEV2:Glu-Thr-Leu-Tyr-Ilue-Gln//Xaa¹ (Xaa = any amino acid; Xaa¹ = any aminoacid, except Pro; // = cleavage site).

EK cleavage sites useful in the present invention include:

Asp-Asp-Asp-Asp-Lys// (SEQ ID NO: 26) (// = cleavage site).

Signal peptides utilized in the present invention may be removed by asignal peptidase or any protease (e.g. Precision, thrombin and factor X)specific for one or more motifs on a signal peptide to generate a matureprotein, including a protein encoded only by the inserted nucleic acid.The present invention also encompasses methods for the production offusion proteins, and the fusion proteins produced by those methods. Inaccordance with the present invention, the proteins of the presentinvention may comprise one or more signal peptides, or portions ofsignal peptides, as noted above. These signal peptides may be used tofacilitate production of desired proteins (e.g. mature or nativeproteins) in vivo or in vitro. Proteins produced using the methods ofthe present invention comprising such signal peptides would allow forthe production of mature proteins, in which proteins are exported fromthe cell upon cleavage of the signal peptide by proteases within thecell. In an in vitro setting, these signal peptides would facilitate theproduction of native or desired proteins outside of a cell. Cleavage ofthe signal peptide may occur using signal peptidases, such as thosedescribed above, thus producing a desired protein product. These signalpeptides may also be used as tags to facilitate affinity purification ofpolypeptides or proteins, for example fusion polypeptides or fusionproteins, produced by the methods of the present invention.

Any number of different protease recognition sites may be used in thepractice of the invention. These sites will often be selected by to fitparticular criteria suitable for the specific application. Exemplaryproteases and protease recognition sites include the following. TobaccoEtch Virus (TEV) protease recognizes the amino acid sequenceGlu-Xaa-Xaa-Tyr-Xaa-Gln//Xaa¹ (SEQ ID NO:23), where Xaa is any aminoacid; Xaa¹ is any amino acid except Pro and // indicates the cleavagesite. Thus, for the amino acid sequence Glu-Asn-Leu-Tyr-Phe-Gln-Gly (SEQID NO:27), TEV cleaves between the Gln and Gly residues (see Invitrogenproduct literature associated with cat. nos. 10127-017 and 12575-015).Also, for the amino acid sequence Glu-Thr-Leu-Tyr-Ile-Gln-Xaa¹ (SEQ IDNO:25), TEV cleaves between the Gln and Xaa residues. Enterokinase (EK)recognizes the amino acid sequence Asp-Asp-Asp-Asp-Lys (SEQ ID NO:26)cleaves after the lysine (see Invitrogen product literature associatedwith cat. nos. E180-01 and E180-02, Invitrogen Corp., Carlsbad, Calif.).The ulp1 protease recognizes the amino acid sequence Gly-Gly-Ser (SEQ IDNO:28) and cleaves between the second glycine and the serine (U.S.Patent Publication No. 2003/0086918). Thus, the invention provides andincludes nucleic acid molecules which may be used for producing proteinswhich may be processed by TEV protease, EK and/or ulp1 protease togenerate proteins, as well as methods employing these enzymes andproteins or peptides produced using these methods.

In instances where the protein or peptide which is desired contains anamino terminal glycine, an amino terminal tag comprising and/or endingin a TEV protease recognition sequence may be used to generate a proteinor peptides which contains no amino acids associated with, for example,cloning sites. Similarly, in instances where the protein which isdesired contains an amino terminal serine, an amino terminal tagcomprising and/or ending in a ulp protease recognition sequence may beused to generate a protein or peptide which contains amino acidsassociated with, for example, cloning sites. EK may be used to generateproteins or peptides which have an amino terminus other than glycine, aswell as glycine.

The present invention also includes methods for joining two or morenucleic acid molecules using methods, for example, described elsewhereherein, wherein a first nucleic acid molecule contains a region whichencodes a protease cleavage site and, optionally, a tag with a secondnucleic acid molecule encodes a desired protein or peptide. In manyinstances, these nucleic acid segments are connected such that thedesired protein is expressed along with amino acids of the proteasecleavage site as a fusion protein such that upon processing with thecognate protease, the desired protein is produced. Often, the desiredprotein which results from proteolytic digestion will contain only aminoacids encoded by the second nucleic acid molecule referred to above.

In many instances, when a desired protein is produced from a nucleicacid formed by the connection of two nucleic acid molecules, thegeneration of a “seam” is only relevant with respect to one end of theprotein (i.e., the amino terminus or the carboxy terminus). In otherwords, in instances, where there is, for example, an amino terminal tagor a carboxy terminal tag, but not both, there is only a need to removeone tag. For example, when the translation product contains an aminoterminal tag, the carboxy terminus of the translation product willtypically terminate at a position in the mRNA which corresponds to thenaturally resident stop codon. In such instances, a protease system maybe used which will only amino terminal amino acids from the translationproduct.

The present invention also encompasses the production of a protein thatcomprises an expression enhancing amino acid sequence cleavable by ulp1protease or an active fragment of ulp1 protease (for example thefragment from amino acid positions 403 to 621) and a poly-amino acid ofinterest, particularly one that is difficult to express in a recombinantexpression system. The protein may also include a purification tag forease of isolation. The ulp1 protease cleavable site may be any ulp1cleavable site, such as for example a ulp1 protease cleavable site froma ubiquitin-like protein e.g. a SUMO (small ubiquitin-like molecule).The SUMO may be, for instance, Smt3 from yeast, or a fragment of Smt3that retains the ability to be recognized and cleaved by Ulp 1. Examplesof such a fragment of Smt3 include the fragment from amino acidpositions 14-98 of Smt3 and the fragment from amino acid positions 1-98of Smt3. Examples of such proteins can be found in WO 02/090495, theentire disclosure of which is incorporated herein by reference.

When nucleic acid molecules and/or methods of the invention are used toproduce proteins or peptides, these proteins or peptides may be producedwith an amino terminal and/or carboxy terminal tag. These tags may beused for any number of purposes, including to (1) increase the stabilityof the protein or peptide or (2) allow for purification. Thus, proteinsor peptides produced by methods of the invention, as well as protein orpeptides encoded by nucleic acid molecules of the invention, may containaffinity purification tags (e.g., epitope tags such as the V5 epitope).Affinity purification tags are often amino acid sequences that caninteract with a binding partner immobilized on a solid support. Nucleicacids encoding multiple consecutive single amino acids, such ashistidine, may be used for one-step purification of the recombinantprotein by affinity binding to a resin column, such as nickel sepharose.A protease cleavage site can be engineered between the affinity tag andthe desired protein to allow for removal of the tag, for example, afterthe purification process is complete or to induce release of the desiredprotein or peptide from the solid support. Affinity tags which may beused in the practice of the invention include tags such as the chitinbinding domain (which binds to chitin), polyarginine,glutathione-S-transferase (which binds to glutathione), maltose bindingprotein (which binds maltose), FlAsH, biotin (which binds to avidin andstrepavidin), and the like.

Epitope tags are short amino acid sequences which are recognized byepitope specific antibodies. Proteins or peptides which contain one ormore epitope tags may purified, for example, using a cognate antibodybound to a chromatography resin. The presence of the epitope tagfurthermore allows the recombinant protein to be detected in subsequentassays, such as Western blots, without having to produce an antibodyspecific for the recombinant protein itself. Examples of commonly usedepitope tags include V5, glutathione-S-transferase (GST), hemaglutinin(HA), the peptide Phe-His-His-Thr-Thr (SEQ ID NO:29), chitin bindingdomain, and the like. As discussed above, these affinity tags may beremoved from the desired protein or peptide by proteolytic cleavage.

FlAsH tags comprise the sequence a cys-cys-Xaa-Xaa-cys-cys (SEQ IDNO:30), where Xaa and Xaa are amino acids. In many instances, Xaa andXaa, which may be the same or different amino acids, are amino acidswith high a-helical propensity. In some embodiments, X and Y are thesame amino acid. These peptides have been shown to bind to biarsenicalcompounds. The FlAsH systems is described in U.S. Pat. No. 6,054,271,the entire disclosure of which is incorporated herein by reference.

The nucleic acid molecules and/or nucleic acid segments utilized in thecloning methods, compositions and kits of the present invention mayoptionally comprise one or more selectable markers comprising at leastone DNA segment encoding an element selected from the group consistingof an antibiotic resistance gene, a gene that encodes a fluorescentprotein, a tRNA gene, an auxotrophic marker, a toxic gene, a phenotypicmarker, an antisense oligonucleotide, a restriction endonuclease, arestriction endonuclease cleavage site, an enzyme cleavage site, aprotein binding site, and a sequence complementary to a PCR primersequence.

Suitable antibiotic resistance genes for use in the present inventionare well known in the art and include, but are not limited to,chloramphenicol resistance genes, ampicillin resistance genes,tetracycline resistance genes, Zeocin resistance genes, spectinomycinresistance genes and kanamycin resistance genes.

Examples of toxic gene products suitable for use in the presentinvention are well known in the art, and include, but are not limitedto, restriction endonucleases (e.g., DpnI), apoptosis-related genes(e.g. ASK1 or members of the bcl-2/ced-9 family), retroviral genesincluding those of the human immunodeficiency virus (HIV), defensinssuch as NP-1, inverted repeats or paired palindromic nucleic acidsequences, bacteriophage lytic genes such as those from (ΦX174 orbacteriophage T4; antibiotic sensitivity genes such as rpsL,antimicrobial sensitivity genes such as pheS, plasmid killer genes,eukaryotic transcriptional vector genes that produce a gene producttoxic to bacteria, such as GATA-1, and genes that kill hosts in theabsence of a suppressing function, e.g., kicB, sacB, ccdB, (ΦX174 E(Liu, Q. et al., Curr. Biol. 8:1300-1309 (1998)), and other genes thatnegatively affect replicon stability and/or replication. The presentinvention also encompasses the use of a gene that encodes the tus genewhich binds to one or more ter sites. A toxic gene can alternatively beselectable in vitro, e.g., a restriction site.

Any of the nucleic acid molecules or nucleic acid segments used in orproduced by the present methods, compositions and kits may furthercomprise one or more site-specific recombination sites. Theserecombination sites may flank the one or more restriction sites (e.g.one or more type IIs sites) if present in the nucleic acid molecules orsegments of the invention. Site-specific recombinases are proteins thatare present in or produced by many organisms (e.g., viruses andbacteria) and have been characterized as having both endonuclease andligase properties. These recombinases (along with associated proteins insome cases) recognize specific sequences of bases (i.e., recombinationsites) in a nucleic acid molecule and exchange the nucleic acid segmentsflanking those sequences. The recombinases and associated proteins arecollectively referred to as “recombination proteins” (see, e.g., Landy,A., Current Opinion in Biotechnology 3:699-707 (1993)).

Numerous recombination systems from various organisms have beendescribed. See, e.g., Hoess, et al., Nucleic Acids Research 14:2287(1986); Abremski, et al., J. Biol. Chem. 261:391 (1986); Campbell, J.Bacteriol. 174:7495 (1992); Qian, et al., J. Biol. Chem. 267:7794(1992); Araki, et al., J. Mol. Biol. 225:25 (1992); Maeser and Kahnmann,Mol. Gen. Genet. 230:170-176) (1991); Esposito, et al., Nucl. Acids Res.25:3605 (1997). Many of these belong to the integrase family ofrecombinases (Argos, et al., EMBO 15:433-440 (1986); Voziyanov, et al.,Nucl. Acids Res. 27:930 (1999)). Perhaps the best studied of these arethe Integrase/att system from bacteriophage ((Landy, A. Current Opinionsin Genetics and Devel. 3:699-707 (1993)), the Cre/loxP system frombacteriophage P1 (Hoess and Abremski (1990) In Nucleic Acids andMolecular Biology, vol. 4. Eds.: Eckstein and Lilley, Berlin-Heidelberg:Springer-Verlag; pp. 90-109), and the FLP/FRT system from theSaccharomyces cerevisiae 2μ circle plasmid (Broach, et al., Cell29:227-234 (1982)).

Recombination sites are sections or segments of nucleic acid on theparticipating nucleic acid molecules that are recognized and bound bythe recombination proteins during the initial stages of integration orrecombination. For example, the recombination site for Cre recombinaseis loxP which is a 34 base pair sequence comprised of two 13 base pairinverted repeats (serving as the recombinase binding sites) flanking an8 base pair core sequence. See FIG. 1 of Sauer, B., Curr. Opin. Biotech.5:521-527 (1994). Other examples of recognition sequences include theattB, attP, attL, and attR sequences which are recognized by therecombination protein Int. attB is an approximately 25 base pairsequence containing two 9 base pair core-type Int binding sites and a 7base pair overlap region, while attP is an approximately 240 base pairsequence containing core-type Int binding sites and arm-type Int bindingsites as well as sites for auxiliary proteins integration host factor(IHF), FIS and excisionase (Xis). See Landy, Curr. Opin. Biotech.3:699-707 (1993). Suitable recombination sites for use in the presentinvention include, but are not limited to, attB sites, attP sites, attLsites, attR sites, lox sites, psi sites, tnpI sites, dif sites, cersites, frt sites, and mutants, variants and derivatives thereof.

The present cloning methods also embody the use of nucleic acidmolecules that include a DNA segment having one or more terminal3′-deoxyadenosine monphosphate (dAMP) residues, as described in U.S.Pat. No. 5,487,933, herein incorporated entirely by reference. These DNAsegments are generated by thermophilic polymerases during PCRamplification. Double-stranded nucleic acids are formed with a singleoverhanging 3′-AMP residue. Mixture of these molecules with a populationof linear double-stranded DNA molecules with a single overhangingdeoxythymidylate (dTMP) residue at one or both of the 3′ termini of theDNA molecule allow for ligation of the 3′-dAMP containing nucleic acidmolecules and the 3′-dTMP-containing DNA molecules to producerecombinant molecules. This approach is commonly known to those in theart as “TA Cloning,” compositions and methods for which are availablefrom Invitrogen Corporation (Carlsbad, Calif.).

The present invention also encompasses the use of cloning methods knownto those skilled in the art as RecA cloning. The RecA cloning proteinefficiently coats singly-stranded DNA. In the presence of ATP, thisRec-A coated single-stranded DNA can for triple-stranded nucleoproteincomplexes with homologous double-stranded DNA. This RecA driven strandinvasion and annealing can lead to high efficiency capture of DNAcontaining regions of homology with single-stranded DNA probes. Thissystem can be used to increase the efficiency of recombination between acircular plasmid DNA molecule and a linear DNA “insert.” Such suitablemethods of RecA cloning can be found in U.S. Pat. Nos. 5,948,653,6,074,853 and 6,200,812, the disclosures of each of which are herebyincorporated entirely by reference.

The present invention also encompasses the use of a method of cloningDNA molecules in cells comprising the steps: a) providing a host cellcapable of performing homologous recombination, b) contacting in saidhost cell a first DNA molecule which is capable of being replicated insaid host cell with a second DNA molecule comprising at least tworegions of sequence homology to regions on the first DNA molecule, underconditions which favour homologous recombination between said first andsecond DNA molecules and c) selecting a host cell in which homologousrecombination between said first and second DNA molecules has occurred.

In this method of the present invention, the homologous recombinationsuitably occurs via the recET mechanism, i.e. the homologousrecombination is mediated by the gene products of the recE and the recTgenes which are preferably selected from the E. coli genes recE and recTor functionally related genes such as the phage λ redα and redβ genes.In contrast to RecA cloning, the recET cloning system requiressignificantly fewer bases of homology for efficient recombination intothe target molecule. These proteins facilitate the homologousincorporation of a double-stranded DNA fragment into a circular plasmid.

A host cell suitable for this embodiment of the present invention is abacterial cell, e.g. a gram-negative bacterial cell. Suitably the hostcell is an enterobacterial cell, such as Salmonella, Klebsielia orEscherichia. Most preferably the host cell is an Escherichia coli cell.It should be noted, however, that this method of the present inventionis also suitable for eukaryotic cells, such as fungi, plant or animalcells. Such suitable methods of recET cloning can be found in Zhang, Y.et al., Nature 20:123-128 (1998), Muryers, J. P. P., et al., Nucl. AcidsRes. 27:1555-1557 (1999), and U.S. Pat. Nos. 6,509,156 and 6,355,412,the disclosures of each of which are hereby incorporated entirely byreference.

The first nucleic acid molecule and/or segment, as well as the secondnucleic acid molecule involved in the methods, compositions and kits ofthe present invention may further or alternatively comprise one or moretopoisomerase recognition sites and/or one or more topoisomerases. Insuitable embodiments, the topoisomerase recognition site(s), if present,may optionally be flanked by two or more recombination sites.

The term “flanked” as used herein is meant to indicate a spatialrelationship wherein a restriction site (e.g. a type IIs site) and/orrecombination site are located to one side of a nucleic acid segment(gene, selectable marker, etc.). As described above, recombination sitesmay also flank restriction sites (e.g. type IIs sites) utilized in theinvention. In the situation where a nucleic acid segment is flanked bytwo or more recombination or recognition sites, each side of the nucleicacid segment may be flanked by one or more sites.

Topoisomerases are categorized as type I, including type IA and type IBtopoisomerases, which cleave a single strand of a double strandednucleic acid molecule, and type II topoisomerases (gyrases), whichcleave both strands of a nucleic acid molecule. Type IA and IBtopoisomerases cleave one strand of a nucleic acid molecule. Cleavage ofa nucleic acid molecule by type IA topoisomerases generates a 5′phosphate and a 3′ hydroxyl at the cleavage site, with the type IAtopoisomerase covalently binding to the 5′ terminus of a cleaved strand.In comparison, cleavage of a nucleic acid molecule by type IBtopoisomerases generates a 3′ phosphate and a 5′ hydroxyl at thecleavage site, with the type IB topoisomerase covalently binding to the3′ terminus of a cleaved strand. The topoisomerase recognition sites ofthe present invention, if present, may be recognized and bound by a typeI topoisomerase, and suitably by a type IB topoisomerase. Type IBtopoisomerases useful in the present invention include, but are notlimited to eukaryotic nuclear type I topoisomerase and a poxvirustopoisomerase. The poxvirus topoisomerase useful in the presentinvention may be produced by or isolated from a virus including, but notlimited to, vaccinia virus, Shope fibroma virus, ORF virus, fowlpoxvirus, molluscum contagiosum virus and Amsacta morrei entomopoxvirus(see Shuman, Biochim. Biophys. Acta 1400:321-337, 1998; Petersen et al.,Virology 230:197-206, 1997; Shuman and Prescott, Proc. Natl. Acad. Sci.,USA 84:7478-7482, 1987; Shuman, J. Biol. Chem. 269:32678-32684, 1994;U.S. Pat. No. 5,766,891; PCT/US95/16099; PCT/US98/12372, each of whichis incorporated herein by reference; see, also, Cheng et al., supra,1998). Suitable type IB topoisomerases include the nuclear type Itopoisomerases present in all eukaryotic cells and those encoded byvaccinia and other cellular poxviruses (see Cheng et al., Cell92:841-850, 1998, which is incorporated herein by reference). Theeukaryotic type IB topoisomerases are exemplified by those expressed inyeast, Drosophila and mammalian cells, including human cells (see Caronand Wang, Adv. Pharmacol. 29B:271-297, 1994; Gupta et al., Biochim.Biophys. Acta 1262:1-14, 1995, each of which is incorporated herein byreference; see, also, Berger, supra, 1998).

In suitable aspects of the present invention, the one or more optionalselectable markers of the nucleic acids or segment used in or producedby the present invention may be flanked by one or more restriction sites(e.g. one or more type IIs sites) and/or one or more recombinationsites.

In other suitable embodiments of the present invention, the firstnucleic acid molecule or segment and/or the second nucleic acid moleculemay not comprise a promoter. The present invention allows for transferof a promoter element into a second nucleic acid molecule that may notcomprise a promoter via seamless cloning. In this orientation,transcription of the second nucleic acid molecule from the promoterelement located on the first nucleic acid molecule may proceed such thatno additional sequences are transcribed between the promoter element andthe start codon of the second nucleic acid molecule. The presentinvention also allows for seamlessly adding a first nucleic acidmolecule or segment into a second nucleic molecule that contains apromoter element such that the first nucleic acid molecule or segmentwill subsequently be under the control of the promoter element.

Incubation conditions suitable for use in the methods of the presentinvention comprise incubation with sufficient amounts of DNA ligases andbuffers. Such incubation conditions are described in Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1982). The term sufficient amount as usedherein means that the amount of DNA ligase(s) and buffer(s) presentduring the cloning and/or recombination reactions is such that thesereactions proceed as designed. Suitable buffers include physiologicbuffers such as, but not limited to,Tris-(hydroxymethyl)aminomethane-HCl TRIS®-HCl,Ethylene-diaminetetraacetic acid (EDTA) disodium salt, saline, PhosphateBuffered Saline (PBS), N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonicacid) (HEPES®), 3-(N-Morpholino)propanesulfonic acid (MOPS),2-bis(2-Hydroxyethylene)amino-2-(hydroxymethyl)-1,3-propanediol(bis-TRIS®), potassium phosphate (KP), sodium phosphate (NaP), dibasicsodium phosphate (Na₂HPO₄), monobasic sodium phosphate (NaH₂PO₄),monobasic sodium potassium phosphate (NaKHPO₄), magnesium phosphate(Mg₃(PO₄)₂.4H₂O), potassium acetate (CH₃COOH), D(+)-α-sodiumglycerophosphate (HOCH₂CH(OH)CH₂OPO₃Na₂) and other physiologic buffersknown to those skilled in the art.

In additional embodiments of the present invention provides methods forcloning or subcloning one or more desired nucleic acid moleculescomprising: (a) combining in vitro or in vivo (i) one or more firstnucleic acid molecules comprising one or more sticky ends generated byone or more first restriction enzymes (e.g. one or more type IIsrestriction enzymes); (ii) one or more second nucleic acid moleculescomprising one or more toxic genes flanked by one or more secondrestriction sites (e.g. one or more type IIs restriction enzymerecognition sites); and (iii) one or more restriction enzymes (e.g. oneor more type IIs restriction enzymes) that are specific for the firstand/or second restriction sites; and (b) incubating the combinationunder conditions sufficient to join the first nucleic acid molecule andone or more of the second nucleic acid molecules, thereby producing oneor more desired product nucleic acid molecules. Cloning via such methodsof the invention allows for selection of successfully cloned nucleicacid molecules where the toxic gene originally present in the secondnucleic acid molecule has been removed and replaced with a desirednucleic acid sequence from the first nucleic acid molecule.

In other embodiments of the present invention provides methods forcloning or subcloning one or more desired nucleic acid molecules, orportions thereof, comprising: (a) combining in vitro or in vivo (i) oneor more first nucleic acid molecules comprising at least one nucleicacid segment that is flanked by one or more first restriction sites(e.g. one or more type IIs restriction enzyme recognition sites); (ii)one or more second nucleic acid molecules comprising one or more toxicgenes flanked by one or more second restriction sites (e.g. one or moretype IIs restriction enzyme recognition sites); and (iii) one or morerestriction enzymes (e.g. one or more type IIs restriction enzymes) thatare specific for the first and/or second restriction enzyme recognitionsites; and (b) incubating the combination under conditions sufficient tojoin the first nucleic acid molecule and one or more of the secondnucleic acid molecules, thereby producing one or more desired productnucleic acid molecules. As noted above, cloning via such methods of theinvention allows for selection of successfully cloned nucleic acidmolecules where the toxic gene originally present in the second nucleicacid molecule has been removed and replaced with a desired nucleic acidsequence from the first nucleic acid molecule.

The present invention also provides methods for cloning or subcloningone or more desired nucleic acid molecules comprising: (a) combining invitro or in vivo (i) one or more first nucleic acid molecules comprisingone or more sticky ends that have been generated by one or more firstrestriction enzymes (e.g. one or more type IIs restriction enzymes);(ii) one or more second nucleic acid molecules comprising one or moretoxic genes and one or more antibiotic resistance genes all flanked byone or more second restriction sites (e.g. one or more type IIsrestriction enzyme recognition sites); and (iii) one or more restrictionenzymes (e.g. one or more type IIs restriction enzymes) that arespecific for the restriction enzyme recognition sites; and (b)incubating said combination under conditions sufficient to join thefirst nucleic acid molecule into and or more of the second nucleic acidmolecules, thereby producing one or more desired product nucleic acidmolecules. This embodiment allows for additional selective screening viaselection, for example, of antibiotic resistant host cells.

The present invention also provides methods for cloning or subcloningone or more desired nucleic acid molecules, or portions thereof,comprising: (a) combining in vitro or in vivo (i) one or more firstnucleic acid molecules comprising at least one nucleic acid segmentflanked by one or more first restriction sites (e.g. one or more typeIIs restriction enzyme recognition sites); (ii) one or more secondnucleic acid molecules comprising one or more toxic genes and one ormore antibiotic resistance genes all flanked by one or more secondrestriction sites (e.g. one or more type IIs restriction enzymerecognition sites); and (iii) one or more restriction enzymes (e.g. oneor more type IIs restriction enzymes) that are specific for therestriction enzyme recognition sites; and (b) incubating saidcombination under conditions sufficient to join the first nucleic acidmolecule and one or more of the second nucleic acid molecules, therebyproducing one or more desired product nucleic acid molecules. Thisembodiment allows for additional selective screening via selection, forexample, of antibiotic resistant host cells.

Another embodiment of the invention provides a method for cloning orsubcloning one or more desired nucleic acid molecules comprising: (a)combining in vitro or in vivo (i) one or more first nucleic acidmolecules comprising one or more sticky ends that have been generated byone or more first restriction enzymes (e.g. one or more type IIsrestriction enzymes); (ii) one or more second nucleic acid moleculescomprising one or more second restriction sites (e.g. one or more typeIIs restriction enzyme recognition sites) flanked by one or morerecombination sites; and (iii) one or more restriction enzymes (e.g. oneor more type IIs restriction enzymes) that are specific for the firstand/or second restriction enzyme recognition sites; and (b) incubatingsaid combination under conditions sufficient to join the first nucleicacid molecule and one or more of said second nucleic acid molecules,thereby producing one or more desired product nucleic acid molecules.Following cloning of the first nucleic acid molecule, the cloned portionof the sequence may be cloned into another nucleic acid molecule via,for example, recombination cloning as described below.

Another embodiment of the invention provides a method for cloning orsubcloning one or more desired nucleic acid molecules, or portionsthereof, comprising: (a) combining in vitro or in vivo (i) one or morefirst nucleic acid molecules comprising at least one nucleic acidsegment flanked by one or more first restriction sites (e.g. one or moretype IIs restriction enzyme recognition sites); (ii) one or more secondnucleic acid molecules comprising one or more second restriction sites(e.g. one or more type IIs restriction enzyme recognition sites) flankedby one or more recombination sites; and (iii) one or more restrictionenzymes (e.g. one or more type IIs restriction enzymes) that arespecific for the first and/or second restriction enzyme recognitionsites; and (b) incubating said combination under conditions sufficientto join the first nucleic acid molecule and one or more of said secondnucleic acid molecules, thereby producing one or more desired productnucleic acid molecules. As noted above, following cloning of the firstnucleic acid molecule, the cloned portion of the sequence may be clonedinto another nucleic acid molecule via, for example, recombinationcloning as described below.

The present invention also provides for a method for cloning orsubcloning one or more desired nucleic acid molecules, or portionsthereof, comprising: (a) combining in vitro or in vivo (i) one or morefirst nucleic acid molecules comprising at least one nucleic acidsegment flanked by one or more first restriction sites (e.g. one or moretype IIs restriction enzyme recognition sites) and further flanked byone or more recombination sites; (ii) one or more second nucleic acidmolecules comprising one or more recombination sites; and (iii) one ormore site-specific recombination proteins; and (b) incubating thecombination under conditions sufficient to transfer the first nucleicacid molecule into one or more of the second nucleic acid molecules,thereby producing one or more desired product nucleic acid molecules.

This method of the present invention allows for the transfer of anucleic acid sequence flanked by one or more restriction sites (e.g. oneor more type IIs sites) that is further flanked by one or morerecombination sites into a second nucleic acid molecule viarecombinational cloning. Recombinational cloning is described in detailin U.S. Pat. Nos. 5,888,732 and 6,277,608 (incorporated herein entirelyby reference in their entireties). Recombinational cloning as disclosedin U.S. Pat. Nos. 5,888,732 and 6,277,608 describes methods for movingor exchanging nucleic acid segments using at least one recombinationsite and at least one recombination protein to provide chimeric DNAmolecules. Suitable recombination proteins for use in the presentinvention include, but are not limited to Int, Cre, IHF, Xis, Fis, Hin,Gin, Cin, Tn3 resolvase, TndX, XerC and XerD.

The methods of the present invention may further comprise introducingthe product nucleic acid into one or more host cells. Host cells thatmay be used in any aspect of the present invention include, but are notlimited to, bacterial cells, yeast cells, plant cells and animal cells.Preferred bacterial host cells include Escherichia spp. cells(particularly E. coli cells and most particularly E. coli strains DH10B,Stb12, DHS, DB3 (deposit No. NRRL B-30098), DB3.1 (preferably E. coliLIBRARY EFFICIENCY₇ DB3.1_(J) Competent Cells; Invitrogen Corporation,Carlsbad, Calif.), DB4 and DB5 (deposit Nos. NRRL B-30106 and NNRLB-30107 respectively, see U.S. application Ser. No. 09/518,188, filedMar. 2, 2000, the disclosure of which is incorporated by referenceherein in its entirety), JDP682 and ccdA-over (See U.S. ProvisionalApplication No. 60/475,004, filed Jun. 3, 2003, the disclosure of whichis incorporated by reference herein in its entirety), Bacillus spp.cells (particularly B. subtilis and B. megaterium cells), Streptomycesspp. cells, Erwinia spp. cells, Klebsiella spp. cells, Serratia spp.cells (particularly S. marcessans cells), Pseudomonas spp. cells(particularly P. aeruginosa cells), and Salmonella spp. cells(particularly S. typhimurium and S. typhi cells). Preferred animal hostcells include insect cells (most particularly Drosophila melanogastercells, Spodoptera frugiperda Sf9 and Sf21 cells and TrichoplusaHigh-Five cells), nematode cells (particularly C. elegans cells), aviancells, amphibian cells (particularly Xenopus laevis cells), reptiliancells, and mammalian cells (most particularly NIH3T3, CHO, COS, VERO,BHK and human cells). Preferred yeast host cells include Saccharomycescerevisiae cells and Pichia pastoris cells. These and other suitablehost cells are available commercially, for example from InvitrogenCorporation (Carlsbad, Calif.), American Type Culture Collection(Manassas, Va.), and Agricultural Research Culture Collection (NRRL;Peoria, Ill.).

Additional host cells that are useful in the present invention includemutant host cells and host cell strains, as well as mutants and/orderivatives thereof, that are resistant to the effects of the expressionof one or more toxic genes. Host cells of this type may, for example,comprise one or more mutations in one or more genes within their genomesor on extrachromosomal or extragenomic DNA molecules (such as plasmids,phagemids, cosmids, etc.), including mutations in, for example, recA,endA, mcrA, mcrB, mcrC, hsd, deoR, tonA, and the like, in particular inrecA or endA or in both recA and endA. The mutations to these host cellsmay render the host cells and host cell strains resistant to toxic genesincluding, but not limited to, ccdB, kicB, sacB, DpnI, anapoptosis-related gene, a retroviral gene, a defensin, a bacteriophagelytic gene, an antibiotic sensitivity gene, an antimicrobial sensitivitygene, a plasmid killer gene, and a eukaryotic transcriptional vectorgene that produces a gene product toxic to bacteria, and mostparticularly ccdB. Production and use of these type of mutant host cellstrains are described in commonly owned U.S. Appl. Nos. 60/122,392,filed Mar. 2, 1999, Ser. No. 09/518,188, filed Mar. 2, 2000 (nowabandoned), 10/396,696, filed Mar. 20, 2003, and 60/475,004, filed Jun.3, 2003, the disclosures of which are incorporated herein by referencein their entireties.

Methods for introducing the cloned product nucleic acid molecules and/orvectors of the invention into the host cells described herein, toproduce host cells comprising one or more of the cloned nucleic acidmolecules and/or vectors of the invention, will be familiar to those ofordinary skill in the art. For instance, the nucleic acid moleculesand/or vectors of the invention may be introduced into host cells usingwell known techniques of infection, transduction, electroporation,transfection, and transformation. The nucleic acid molecules and/orvectors of the invention may be introduced alone or in conjunction withother the nucleic acid molecules and/or vectors and/or proteins,peptides or RNAs. Alternatively, the nucleic acid molecules and/orvectors of the invention may be introduced into host cells as aprecipitate, such as a calcium phosphate precipitate, or in a complexwith a lipid. Electroporation also may be used to introduce the nucleicacid molecules and/or vectors of the invention into a host. Likewise,such molecules may be introduced into chemically competent cells such asE. coli. If the vector is a virus, it may be packaged in vitro orintroduced into a packaging cell and the packaged virus may betransduced into cells. Hence, a wide variety of techniques suitable forintroducing the nucleic acid molecules and/or vectors of the inventioninto cells in accordance with this aspect of the invention are wellknown and routine to those of skill in the art. Such techniques arereviewed at length, for example, in Sambrook, J., et al., MolecularCloning, a Laboratory Manual, 2nd Ed., Cold Spring Harbor, N.Y.: ColdSpring Harbor Laboratory Press, pp. 16.30-16.55 (1989), Watson, J. D.,et al., Recombinant DNA, 2nd Ed., New York: W.H. Freeman and Co., pp.213-234 (1992), and Winnacker, E.-L., From Genes to Clones, New York:VCH Publishers (1987), which are illustrative of the many laboratorymanuals that detail these techniques and which are incorporated byreference herein in their entireties for their relevant disclosures.

The present invention also encompasses producing a subsequent nucleicacid and/or a protein by introduction of a cloned product nucleic acidmolecule of the invention and expression in a host cell. Methods andconditions by which to produce such product nucleic acid molecules andproduct proteins are well known in the art. See for example, Sambrook,J., et al., Molecular Cloning, a Laboratory Manual, 2nd Ed., Cold SpringHarbor, N.Y.: Cold Spring Harbor Laboratory Press (1989).

The present invention also encompasses the nucleic acid molecules andproteins produced from a host cell of the invention. An improvement ofthe present invention is that nucleic acid molecules produced usingmethods of the present invention, in many instances, will not containextraneous nucleotides that are not associated with the desired nucleicacid, for example nucleotides encoded by the restriction sites (e.g.type IIs restriction enzyme recognition sites). In other words, theseamless cloning methods of the present invention allow for a productmolecule that does not contain extraneous nucleotides from othersources, including the restriction sites. Similarly, the product proteinmolecules produced using the methods of the present invention are freeof amino acids that are not associated with the desired native or matureproduct protein, for example the product protein molecules are free ofamino acids encoded by the restriction sites (e.g. type IIs restrictionsites). The proteins produced by the methods of the invention may be ofany size, including for example, a short peptide from about 5 aminoacids, about 10 amino acids, about 20 amino acids, about 30 amino acids,about 40 amino acids, about 50 amino acids. The present invention alsoencompasses the production of larger proteins, for example about 300amino acids in length, or even a large protein of greater than about 600amino acids in length.

In one embodiment of the present invention, the nucleic acid moleculesproduced from the host cells may be useful as interfering RNA molecules.In biological systems that are not amenable to gene targeting orhomologous recombination, a process called RNA interference (RNAi) isone practical method of generating knockout (KO) phenotypes. Posttranscriptional gene silencing (PTGS) in plants and quelling inNeurospora was described in the early 1990s. RNAi was originallydescribed in the model organism C. elegans as double stranded RNA(dsRNA) that mediated sequence specific gene silencing (Fire et al.,Nature 391:806-811 (1998)). RNAi has also been described in yeast,Drosophila, plants and trypanosomes. RNAi can be used for geneticanalysis. For example, it can be used for genome wide RNAi screens. RNAihas been shown to be conserved in mammals. RNAi has been used in theidentification of a short interfering RNA (siRNA) as an effectormolecule and with microRNA (miRNA) regulation. Essentially, the processinvolves application of double stranded RNA (dsRNA) that represents acomplementary sense and anti-sense strand of a portion of a target genewithin the region that encodes mRNA. The presence of the interferingdsRNA causes a severe post-transcriptional down-regulation of the targetgene. This versatile technique has been used as a tool in the study ofeukaryotic biology (see Sharp, P. A., Genes Dev. 13:139-141 (1999)).RNAi is an evolutionarily conserved phenomenon and a multi-step processthat involves generation of active small interfering RNA (siRNA) in vivothrough the action of an RNase III endonuclease, DICER, which digestslong double stranded RNA molecules (dsRNA) into shorter fragments (SeeFIG. 13). The 21- to 23-nucleotide base pair small interfering RNAs(siRNAs), produced through the action of DICER, mediate degradation ofthe complementary homologous RNA. One bottleneck to using RNAi as a toolhas been mRNA target site selection. Yet another challenge has beendelivery, either transient such as transfection of dsRNA (See FIGS.16-18) (Kawasaki et. al, NAR, 31(3):981-987 (2003)) or stable expressionusing vectors or a virus (See FIGS. 15 and 19) (Dykxhoorn, Novina andSharp, Nature Reviews, Vol. 4, (June 2003)). RNAi has successfully beenreported in stable cell lines and transgenic mice. GFP shRNA block GFPexpression in transgenic mice, decrease GFP in blastocytes and lower GFPfluorescence overall in a three day pup with two copies of the shRNA(Tiscornia et. al, PNAS, 2003).

RNAi is also powerful in reverse genetics. RNAi can be used as a loss offunction tool, similar to antisense and ribozymes, but more potent.Natural cellular machinery use double stranded RNA to regulate cellularprocesses (e.g., miRNA). Some advantages of RNAi are that it is broadlyconserved in eukaryotic organisms, is post transciptional (effective indiploids) and is tunable (can adjust level of RNAi at several levels).

Until recently, RNAi technology did not appear to be applicable tomammalian systems. In mammals, dsRNA activates dsRNA-activated proteinkinase (PKR) resulting in an apoptotic cascade and cell death (Der etal, Proc. Natl. Acad. Sci. USA 94:3279-3283 (1997)). In addition, it haslong been known that dsRNA activates the interferon cascade in mammaliancells, which can also lead to altered cell physiology (Colby et al,Annu. Rev. Microbiol. 25:333 (1971); Kleinschmidt et al., Annu. Rev.Biochem. 41:517 (1972); Lampson et al., Proc. Natl. Acad. Sci. USA58L782 (1967); Lomniczi et al., J. Gen. Virol. 8:55 (1970); Younger etal., J. Bacteriol. 92:862 (1966)). However, dsRNA-mediated activation ofthe PKR and interferon cascades typically require dsRNA longer thanabout 30 base pairs. Since the primary products of DICER are 21-23 basepair fragments of dsRNA, one can circumvent the adverse or undesiredmammalian responses to dsRNA and still elicit an interfering RNA effectvia siRNA (Elbashir et al., Nature 411:494-498 (2001)).

Thus, another aspect of the present invention provides methods ofproducing an RNA molecule for use as an interfering RNA comprising: (a)optionally, identifying one or more target nucleic acid sequences; (b)preparing one or more nucleic acid molecules which encode one or moreinterfering RNAs, wherein the interfering RNAs bind to the one or moretarget nucleic acid sequences; (c) combining in vitro or in vivo, (i)the one or more first nucleic acid molecules encoding one or moreinterfering RNAs that have one or more sticky ends that have beengenerated by one or more restriction enzymes (e.g. type IIs restrictionenzymes); and (ii) one or more second nucleic acid molecules comprisingone or more ends which are compatible with the one or more sticky endson the first nucleic acid molecule(s), and optionally comprising one ormore selectable markers; and (d) incubating the combination underconditions sufficient to join one or more of the nucleic acid moleculesencoding the interfering RNAs and one or more of the second nucleic acidmolecules, thereby producing one or more desired product nucleic acidmolecules; (e) inserting the one or more product nucleic acid moleculesinto a host cell; and (f) expressing the one or more interfering RNAs inthe host cell.

The present invention also provides methods of producing an RNA moleculefor use as an interfering RNA comprising: (a) optionally, identifyingone or more target nucleic acid sequences; (b) preparing one or morenucleic acid molecules which encode one or more interfering RNAs,wherein the interfering RNAs bind to the one or more target nucleic acidsequences; (c) combining in vitro or in vivo, (i) the one or more firstnucleic acid molecules encoding one or more interfering RNAs flanked byone or more first restriction sites (e.g. one or more type IIsrestriction enzyme recognition sites); (ii) one or more second nucleicacid molecules comprising one or more second restriction sites (e.g. oneor more type IIs restriction enzyme recognition sites) and optionallycomprising one or more selectable markers; and (iii) one or moresite-specific restriction enzymes (e.g. one or more type IIs restrictionenzymes); and (d) incubating the combination under conditions sufficientto join one or more of the nucleic acid molecules encoding theinterfering RNAs and one or more of the second nucleic acid molecules,thereby producing one or more desired product nucleic acid molecules;(e) inserting the one or more product nucleic acid molecules into a hostcell; and (f) expressing the one or more interfering RNAs in the hostcell.

In yet another embodiment, the present invention provides methods ofproducing an RNA molecule for use as an interfering RNA comprising: (a)optionally, identifying one or more target nucleic acid sequences; (b)preparing one or more nucleic acid molecules which encode one or moreinterfering RNAs, wherein the interfering RNAs bind to the one or moretarget nucleic acid sequences; (c) combining in vitro or in vivo, (i)the one or more first nucleic acid molecules encoding one or moreinterfering RNAs that have one or more sticky ends that have beengenerated by one or more restriction enzymes (e.g. type IIs restrictionenzymes); and (ii) one or more second nucleic acid molecules comprisingone or more ends which are compatible with the one or more sticky endson the first nucleic acid molecule(s), and optionally comprising one ormore selectable markers; and (d) incubating the combination underconditions sufficient to join one or more of the nucleic acid moleculesencoding the interfering RNAs and one or more of the second nucleic acidmolecules, thereby producing one or more desired product nucleic acidmolecules; and (e) expressing one or more interfering RNAs in vitro orin vivo. In a first further embodiment, the one or more interfering RNAsmay be produced in vitro or isolated from a cell and then introducedinto a second cell.

Another aspect of the present invention provides methods of producing anRNA molecule for use as an interfering RNA comprising: (a) optionally,identifying one or more target nucleic acid sequences; (b) preparing oneor more nucleic acid molecules which encode one or more interferingRNAs, wherein the interfering RNAs bind to the one or more targetnucleic acid sequences; (c) combining in vitro or in vivo, (i) the oneor more first nucleic acid molecules encoding one or more interferingRNAs flanked by one or more first restriction sites (e.g. one or moretype IIs restriction enzyme recognition sites); (ii) one or more secondnucleic acid molecules comprising one or more second restriction sites(e.g. one or more type IIs restriction enzyme recognition sites) andoptionally comprising one or more selectable markers; and (iii) one ormore site-specific restriction enzymes (e.g. one or more type IIsrestriction enzymes); and (d) incubating the combination underconditions sufficient to join one or more of the nucleic acid moleculesencoding the interfering RNAs and one or more of the second nucleic acidmolecules, thereby producing one or more desired product nucleic acidmolecules; and (e) expressing one or more interfering RNAs in vitro orin vivo. In a first further embodiment, the one or more interfering RNAsmay be produced in vitro or isolated from a cell and then introducedinto a second cell.

Another aspect of the present invention provides methods of producing anRNA molecule for use as an interfering RNA comprising: (a) optionally,identifying one or more target nucleic acid sequences; (b) preparing oneor more interfering RNAs, wherein the interfering RNAs bind to the oneor more target nucleic acid sequences; (c) combining in vitro or invivo, (i) the one or more first nucleic acid molecules comprising one ormore interfering RNAs that have one or more sticky ends that have beengenerated by one or more restriction enzymes (e.g. type IIs restrictionenzymes); and (ii) one or more second nucleic acid molecules comprisingone or more ends which are compatible with the one or more sticky endson the first nucleic acid molecule(s), and optionally comprising one ormore selectable markers; and (d) incubating the combination underconditions sufficient to join one or more interfering RNAs and one ormore of the second nucleic acid molecules, thereby producing one or moredesired product nucleic acid molecules; (e) inserting the one or moreproduct nucleic acid molecules into a host cell; and (f) expressing theone or more interfering RNAs in the host cell.

The present invention also provides methods of producing an RNA moleculefor use as an interfering RNA comprising: (a) optionally, identifyingone or more target nucleic acid sequences; (b) preparing one or morenucleic acid molecules which comprise one or more interfering RNAs,wherein the interfering RNAs bind to the one or more target nucleic acidsequences; (c) combining in vitro or in vivo, (i) the one or more firstnucleic acid molecules comprising one or more interfering RNAs flankedby one or more first restriction sites (e.g. one or more type IIsrestriction enzyme recognition sites); (ii) one or more second nucleicacid molecules comprising one or more second restriction sites (e.g. oneor more type IIs restriction enzyme recognition sites) and optionallycomprising one or more selectable markers; and (iii) one or moresite-specific restriction enzymes (e.g. one or more type IIs restrictionenzymes); and (d) incubating the combination under conditions sufficientto join one or more interfering RNAs and one or more of the secondnucleic acid molecules, thereby producing one or more desired productnucleic acid molecules; (e) inserting the one or more product nucleicacid molecules into a host cell; and (f) expressing the one or moreinterfering RNAs in the host cell.

Suitable nucleic acid molecules that can function as interfering RNA(iRNA) and that can be produced using the methods of the presentinvention may be either single- or double-stranded RNA (ssRNA or dsRNA,respectively). Examples of iRNA produced via methods of the presentinvention include, but are not limited to, antisense oligonucleotides,ribozymes, small interfering RNAs, double stranded RNAs, invertedrepeats, short hairpin RNAs, small temporally regulated RNAs and thelike.

Antisense Oligonucleotides

In general, antisense oligonucleotides comprise one or more nucleotidesequences sufficient in identity, number and size to effect specifichybridization with a preselected nucleic. Antisense oligonucleotidesproduced in accordance with the present invention typically havesequences that are selected to be sufficiently complementary to thetarget nucleic sequences (suitably mRNA in a target cell or organism) sothat the antisense oligonucleotide forms a stable hybrid with the mRNAand inhibits the translation of the mRNA sequence, preferably underphysiological conditions. It is preferred but not necessary that theantisense oligonucleotide be 100% complementary to a portion of thetarget gene sequence. However, the present invention also encompassesthe production of antisense oligonucleotides with a different level ofcomplementarity to the target gene sequence, e.g., antisenseoligonucleotides that are at least about 50% complementary, at leastabout 55% complementary, at least about 60% complementary, at leastabout 65% complementary, at least about 70% complementary, at leastabout 75% complementary, at least about 80% complementary, at leastabout 85% complementary, at least about 90% complementary, at leastabout 91% complementary, at least about 92% complementary, at leastabout 93% complementary, at least about 94% complementary, at leastabout 95% complementary, at least about 96% complementary, at leastabout 97% complementary, at least about 98% complementary, or at leastabout 99% complementary, to the target gene sequence.

Antisense oligonucleotides that may be produced in accordance with thepresent invention are well known in the art and that will be familiar tothe ordinarily skilled artisan. Representative teachings regarding thesynthesis, design, selection and use of antisense oligonucleotidesinclude without limitation U.S. Pat. No. 5,789,573, U.S. Pat. No.6,197,584, and Ellington, “Current Protocols in Molecular Biology,” 2ndEd., Ausubel et al., eds., Wiley Interscience, New York (1992), thedisclosures of which are incorporated by reference herein in theirentireties.

Ribozymes

In general, ribozymes are RNA molecules having enzymatic activitiesusually associated with cleavage, splicing or ligation of nucleic acidsequences to which the ribozyme binds. Typical substrates for ribozymesinclude RNA molecules, although ribozymes may also catalyze reactions inwhich DNA molecules serve as substrates. Two distinct regions can beidentified in a ribozyme: the binding region which gives the ribozymeits specificity through hybridization to a specific nucleic acidsequence, and a catalytic region which gives the ribozyme the activityof cleavage, ligation or splicing. Ribozymes which are activeintracellularly work in cis, catalyzing only a single turnover, and areusually self-modified during the reaction. However, ribozymes can beengineered to act in trans, in a truly catalytic manner, with a turnovergreater than one and without being self-modified. Owing to the catalyticnature of the ribozyme, a single ribozyme molecule cleaves manymolecules of target nucleic acids and therefore therapeutic activity isachieved in relatively lower concentrations than those required in anantisense treatment (WO 96/23569).

Ribozymes that may be produced in accordance with the present inventionare well known in the art and that will be familiar to the ordinarilyskilled artisan. Representative teachings regarding the synthesis,design, selection and use of ribozymes include without limitation U.S.Pat. No. 4,987,071, and U.S. Pat. No. 5,877,021, the disclosures of allof which are incorporated herein by reference in their entireties.

Small Interfering RNAs (siRNA)

RNAi is mediated by double stranded RNA (dsRNA) molecules that havesequence-specific homology to their “target” nucleic acid sequences(Caplen, N. J., et al., Proc. Natl. Acad. Sci. USA 98:9742-9747 (2001)).Biochemical studies in Drosophila cell-free lysates indicate that, incertain embodiments of the present invention, the mediators ofRNA-dependent gene silencing are 21-25 nucleotide “small interfering”RNA duplexes (siRNAs). Accordingly, siRNA molecules are suitably used inmethods of the present invention. The siRNAs are derived from theprocessing of dsRNA by an RNase known as Dicer (Bernstein, E., et al.,Nature 409:363-366 (2001)). It appears that siRNA duplex products arerecruited into a multi-protein siRNA complex termed RISC (RNA InducedSilencing Complex). Without wishing to be bound by any particulartheory, a RISC is then believed to be guided to a target nucleic acid(suitably mRNA), where the siRNA duplex interacts in a sequence-specificway to mediate cleavage in a catalytic fashion (Bernstein, E., et al.,Nature 409:363-366 (2001); Boutla, A., et al., Curr. Biol. 11:1776-1780(2001)).

Small interfering RNAs that may be produced in accordance with thepresent invention are well known in the art and that will be familiar tothe ordinarily skilled artisan. Small interfering RNAs that may beproduced via the methods of the present invention suitably comprisebetween about 1 to about 50 nucleotides (nt). For example, siRNAs maycomprise about 5 to about 40 nt, about 5 to about 30 nt, about 10 toabout 30 nt, or about 15 to about 30 nt. Longer siRNAs (greater thanabout 30 nucleotides in length) may be useful in some non-human animalsystems, and may suitably be produced by the methods of the presentinvention. Most reports describe the use of U6 or H1 pol III promotersto drive production of siRNA (Lee et al., Nat. Biotechnol. 20:500-505(2002); Paddison et al., Genes Dev. 16:948-958 (2002); Brummelkamp etal., Science 296:550-553 (2002)). Pol III promoters have all theelements required for initiation of transcription upstream of a definedtranscription start site and terminate transcription at 4 or more Ts(incorporating only 1 or 2 Us into the 3′ end of the nascent RNA). Theseattributes allow the production of short RNA molecules with definedends.

Inverted Repeats

Inverted repeats comprise single stranded nucleic acid molecules thatcontain two sequences complementary to each other, oriented such thatone of the sequences is inverted relative to the other. This orientationallows the two complementary sequences to base pair with each other,thereby forming a hairpin structure. The two copies of the invertedrepeat need not be contiguous. There may be “n” additional nucleotidesbetween the hairpin forming sequences, wherein “n” is any number ofnucleotides. For example, n can be about 1, about 5, about 10, about 50,or about 100 nucleotide, or more, and can be any number of nucleotidesfalling within these discrete values.

Inverted repeats suitable that may be produced in accordance with thepresent invention can be synthesized and used according to proceduresthat are well known in the art and that will be familiar to theordinarily skilled artisan. The production and use of inverted repeatsfor RNA interference can be found in, without limitation, Kirby, K., etal., Proc. Natl. Acad. Sci. USA 99:16162-16167 (2002), Adelman, Z. N.,et al., J. Virol. 76:12925-12933 (2002), Yi, C. E., et al., J. Biol.Chem. 278:934-939 (2003), Yang, S., et al., Mol. Cell Biol. 21:7807-7816(2001), Svoboda, P., et al., Biochem. Biophys. Res. Commun.287:1099-1104 (2001), and Martinek, S, and Young, M. W., Genetics156:171-1725 (2000).

Short Hairpin RNA (shRNA)

Paddison, P. J., et al., Genes & Dev. 16:948-958 (2002) have used smallRNA molecules folded into hairpins as a means to effect RNAi.Accordingly, such short hairpin RNA (shRNA) molecules that may beproduced via the methods of the present invention. Functionallyidentical to the inverted repeats described herein, the length of thestem and loop of functional shRNAs distinguishes them from invertedrepeats. Stem lengths can range from about 1 to about 30 nt, and loopsize can range between 1 to about 25 nt without affecting silencingactivity. While not wishing to be bound by any particular theory, it isbelieved that these shRNAs resemble the dsRNA products of the DicerRNase and, in any event, have the same capacity for inhibitingexpression of a specific gene.

Transcription of shRNAs is initiated at a polymerase III (pol III)promoter (e.g. U6 and H1 promoters) and is believed to be terminated atposition 2 of a 4-5-thymine transcription termination site. Uponexpression, shRNAs are thought to fold into a stem-loop structure with3′ UU-overhangs. Subsequently, the ends of these shRNAs are processed,converting the shRNAs into ˜21 nt siRNA-like molecules.

Short hairpin RNAs that may be produced in accordance with the presentinvention are well known in the art and that will be familiar to theordinarily skilled artisan. The production and use of inverted repeatsfor RNA interference can be found in, without limitation, Paddison, P.J., et al., Genes & Dev. 16:948-958 (2002), Yu, J-Y., et al. Proc. Natl.Acad. Sci. USA 99:6047-6052 (2002), and Paul, C. P. et al. NatureBiotechnol. 20:505-508 (2002).

MicroRNAs (miRNAs)

The invention may further be used to produce microRNA molecules.MicroRNA molecules are molecules which are structurally similar to shRNAmolecules but, typically, contain one or more mismatches orinsertion/deletions in their regions of sequence complementary. Hundredsof miRNAs have been identified in C. elegans, flies and humans. C.elegans miRNA, lin-4 and let-7, have been identified to regulatedevelopmental timing and inhibit expression of targeted genes. Examplesof miRNA regulation from yeast to humans includes regulation ofchromatin structure in yeast and tumor suppressor genes in humans. Atleast some microRNA molecules are transcribed as polycistrons of about400, which are then processed to RNA molecules of about 70 nucleotides.These double stranded 70 mers are then processed again, presumably bythe enzyme Dicer, to two RNA molecules which are about 22 nucleotides inlength and often have one or more (e.g., one, two, three, four, five,etc.) internal mismatches in their regions of sequence complementarity.(See FIG. 25) (Lee et al., EMBO 21:4663-4670 (2002). The miRNA can entera miRNA ribonucleoprotein particle (miRNP) similar to siRNA enteringinto the RISC protein complex (FIG. 14) (Dykxhoorn, Novina and Sharp,Nature Reviews, Vol. 4, (June 2003)). The binding of miRNA/siRNAs ofperfect complementarity to a target results in mRNA degradation; singlebase mismatches can block translation. The invention also includes, forexample, uses of microRNA molecules and nucleic acid molecules whichencode microRNA molecules which are similar to the uses described hereinfor shRNA and non-hairpin double stranded RNA molecules.

Small Temporally Regulated RNAs (stRNAs)

Another group of small RNAs that may be produced via the methods of thepresent invention are the small temporally regulated RNAs (stRNAs). Ingeneral, stRNAs comprise from about 20 to about 30 nt (Banerjee andSlack, Bioessays 24:119-129 (2002)), although stRNAs of any size arealso suitable for use in accordance with the invention. Unlike siRNAs,stRNAs downregulate expression of a target mRNA after the initiation oftranslation without degrading the mRNA.

The nucleic acids used in accordance with the present invention can beconveniently and routinely made through the well-known technique ofsolid-phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Other methods for such synthesis that are known in the art mayadditionally or alternatively be employed. It is well-known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives. By way of non-limitingexample, see, e.g., U.S. Pat. Nos. 4,517,338, and 4,458,066; Lyer R P,et al., Curr. Opin. Mol. Ther. 1:344-358 (1999); and Verma S, andEckstein F., Annual Rev. Biochem. 67:99-134 (1998), the disclosures ofall of which are incorporated herein by reference in their entireties.

The present invention also provides methods for the production of geneknockout/knockdown cells and cells lines, as well as geneticallymodified transgenic animals.

In such suitable embodiments, the present invention provides methods ofregulating the expression of one or more genes in a cell or an animalusing interfering RNA, comprising: (a) identifying one or more targetnucleic acid sequences; (b) preparing one or more nucleic acid moleculeswhich encode one or more interfering RNAs, wherein the interfering RNAsbind to the one or more target nucleic acid sequences; (c) combining invitro or in vivo, (i) the one or more first nucleic acid moleculesencoding one or more interfering RNAs that have one or more sticky endsthat have been generated by one or more restriction enzymes (e.g. typeIIs restriction enzymes); and (ii) one or more second nucleic acidmolecules comprising one or more ends which are compatible with the oneor more sticky ends on the first nucleic acid molecule(s), andoptionally comprising one or more selectable markers; (d) incubating thecombination under conditions sufficient to join one or more of thenucleic acid molecules encoding the interfering RNAs and one or more ofthe second nucleic acid molecules, thereby producing one or more desiredproduct nucleic acid molecules; and (e) inserting the one or moreinterfering RNA expression vectors into the cell or one or more cells ofthe animal, under conditions such that the one or more interfering RNAsbind to the one or more target nucleic acid sequences, therebyregulating expression of the one or more targeted genes.

The related embodiments, the present invention also provides methods ofregulating the expression of one or more genes in a cell or an animalusing interfering RNA, comprising: (a) identifying one or more targetnucleic acid sequences; (b) preparing one or more nucleic acid moleculeswhich comprise one or more interfering RNAs, wherein the interferingRNAs bind to the one or more target nucleic acid sequences; (c)combining in vitro or in vivo, (i) the one or more first nucleic acidmolecules comprising one or more interfering RNAs flanked by one or morefirst restriction sites (e.g. one or more type IIs restriction enzymerecognition sites); (ii) one or more second nucleic acid moleculescomprising one or more second restriction sites (e.g. one or more typeIIs restriction enzyme recognition sites) and optionally comprising oneor more selectable markers; and (iii) one or more site-specificrestriction enzymes (e.g. one or more type IIs restriction enzymes); (d)incubating the combination under conditions sufficient to join one ormore interfering RNAs and one or more of the second nucleic acidmolecules, thereby producing one or more desired product nucleic acidmolecules; and (e) inserting the one or more interfering RNA expressionvectors into the cell or one or more cells of the animal, underconditions such that the one or more interfering RNAs bind to the one ormore target nucleic acid sequences, thereby regulating expression of theone or more targeted genes.

The nucleic acid molecules of the invention can also be used to producetransgenic organisms (e.g., animals and plants). Animals of any species,including, but not limited to, mice, rats, rabbits, hamsters, guineapigs, pigs, micro-pigs, goats, sheep, cows and non-human primates (e.g.,baboons, monkeys, and chimpanzees) may be used to generate transgenicanimals. Further, plants of any species, including but not limited toLepidium sativum, Brassica juncea, Brassica oleracea, Brassica rapa,Acena sativa, Triticum aestivum, Helianthus annuus, Colonial bentgrass,Kentucky bluegrass, perennial ryegrass, creeping bentgrass,Bermudagrass, Buffalograss, centipedegrass, switch grass, Japaneselawngrass, coastal panicgrass, spinach, sorghum, tobacco and corn, maybe used to generate transgenic plants.

Any technique known in the art may be used to introduce nucleic acidmolecules of the invention into organisms to produce the founder linesof transgenic organisms. Such techniques include, but are not limitedto, pronuclear microinjection (Paterson et al., Appl. Microbiol.Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology (NY)11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834 (1991);and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirus mediatedgene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad.Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; gene targetingin embryonic stem cells (Thompson et al., Cell 56:313-321 (1989));electroporation of cells or embryos (Lo, Mol. Cell. Biol. 3:1803-1814(1983)); introduction of the polynucleotides of the invention using agene gun (see, e.g., Ulmer et al., Science 259:1745 (1993); introducingnucleic acid constructs into embryonic pluripotent stem cells andtransferring the stem cells back into the blastocyst; and sperm-mediatedgene transfer (Lavitrano et al., Cell 57:717-723 (1989); etc. For areview of such techniques, see Gordon, “Transgenic Animals,” Intl. Rev.Cytol. 115:171-229 (1989), which is incorporated by reference herein inits entirety. Further, the contents of each of the documents recited inthis paragraph is herein incorporated by reference in its entirety. Seealso, U.S. Pat. No. 5,464,764 (Capecchi et al., Positive-NegativeSelection Methods and Vectors); U.S. Pat. No. 5,631,153 (Capecchi etal., Cells and Non-Human Organisms Containing Predetermined GenomicModifications and Positive-Negative Selection Methods and Vectors forMaking Same); U.S. Pat. No. 4,736,866 (Leder et al., TransgenicNon-Human Animals); and U.S. Pat. No. 4,873,191 (Wagner et al., GeneticTransformation of Zygotes); each of which is hereby incorporated byreference in its entirety.

Any technique known in the art may be used to produce transgenic clonescontaining nucleic acid molecules of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)), each ofwhich is herein incorporated by reference in its entirety).

The present invention provides for transgenic organisms that carrynucleic acid molecules of the invention in all their cells, as well asorganisms which carry these nucleic acid molecules, but not all theircells, i.e., mosaic organisms or chimeric. The nucleic acid molecules ofthe invention may be integrated as a single copy or as multiple copiessuch as in concatamers, e.g., head-to-head tandems or head-to-tailtandems. The nucleic acid molecules of the invention may also beselectively introduced into and activated in a particular cell type byfollowing, for example, the teaching of Lasko et al. (Lasko et al.,Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)). The regulatorysequences required for such a cell-type specific activation will dependupon the particular cell type of interest, and will be apparent to thoseof skill in the art. When it is desired that nucleic acid molecules ofthe invention be integrated into the chromosomal site of the endogenousgene, this will normally be done by gene targeting. Briefly, when such atechnique is to be utilized, vectors containing some nucleotidesequences homologous to the endogenous gene are designed for the purposeof integrating, via homologous recombination with chromosomal sequences,into and disrupting the function of the nucleotide sequence of theendogenous gene. Nucleic acid molecules of the invention may also beselectively introduced into a particular cell type, thus inactivatingthe endogenous gene in only that cell type, by following, for example,the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). Theregulatory sequences required for such a cell-type specific inactivationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art. The contents of each of thedocuments recited in this paragraph is herein incorporated by referencein its entirety.

Once transgenic organisms have been generated, the expression of therecombinant gene may be assayed utilizing standard techniques. Initialscreening may be accomplished by Southern blot analysis or PCRtechniques to analyze organism tissues to verify that integration ofnucleic acid molecules of the invention has taken place. The level ofmRNA expression of nucleic acid molecules of the invention in thetissues of the transgenic organisms may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the organism, in situ hybridizationanalysis, and reverse transcriptase-PCR (rt-PCR). Samples of tissue maywhich express nucleic acid molecules of the invention also be evaluatedimmunocytochemically or immunohistochemically using antibodies specificfor the expression product of these nucleic acid molecules.

Once the founder organisms are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular organism.Examples of such breeding strategies include, but are not limited to:outbreeding of founder organisms with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenic organisms that express nucleic acidmolecules of the invention at higher levels because of the effects ofadditive expression of each copy of nucleic acid molecules of theinvention; crossing of heterozygous transgenic organisms to produceorganisms homozygous for a given integration site in order to bothaugment expression and eliminate the need for screening of organisms byDNA analysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the nucleic acidmolecules of the invention on a distinct background that is appropriatefor an experimental model of interest.

Transgenic and “knock-out” organisms of the invention have uses whichinclude, but are not limited to, model systems (e.g., animal modelsystems) useful in elaborating the biological function of expressionproducts of nucleic acid molecules of the invention, studying conditionsand/or disorders associated with aberrant expression of expressionproducts of nucleic acid molecules of the invention, and in screeningfor compounds effective in ameliorating such conditions and/ordisorders.

As one skilled in the art would recognize, in many instances whennucleic acid molecules of the invention are introduced into metazoanorganisms, it will be desirable to operably link sequences which encodeexpression products to tissue-specific transcriptional regulatorysequences (e.g., tissue-specific promoters) where production of theexpression product is desired. Such promoters can be used to facilitateproduction of these expression products in desired tissues. Aconsiderable number of tissue-specific promoters are known in the art.Further, methods for identifying tissue-specific transcriptionalregulatory sequences are described elsewhere herein.

The present invention also provides isolated nucleic acids comprising:(a) one or more sticky ends that have been generated by one or morerestriction enzymes (e.g. one or more type IIs restriction enzymes); and(b) optionally one or more selectable markers. The present inventionfurther provides isolated nucleic acids comprising: (a) one or morerestriction sites (e.g. one or more type IIs restriction enzymerecognition sites); and (b) optionally one or more selectable markers.As noted above, selectable markers for use in the isolated nucleic acidsof the present invention comprise antibiotic resistance genes and toxicgenes. As also described above, the isolated nucleic acids molecules ofthe present invention may also comprise one or more recombination sites,and one or more topoisomerase recognition sites and/or one or moretopoisomerases. In suitable embodiments, the topoisomerase recognitionsite, if present, may optionally be flanked by two or more recombinationsites.

In another embodiment, the present invention provides isolated nucleicacids comprising: (a) one or more sticky ends that have been generatedby one or more restriction enzymes (e.g. one or more type IIsrestriction enzymes); and (b) one or more recombination sites. In yetanother embodiment, the present invention provides isolated nucleicacids comprising: (a) one or more restriction sites (e.g. one or moretype IIs restriction enzyme recognition sites); and (b) one or morerecombination sites. Suitable recombination sites include, but are notlimited to, attB sites, attP sites, attL sites, attR sites, lox sites,psi sites, tnpI sites, dif sites, cer sites, frt sites, and mutants,variants and derivatives thereof. In suitable embodiments, the isolatednucleic acid molecules of the present invention may optionally compriseone or more selectable markers, one or more topoisomerase recognitionsites and/or one or more topoisomerases. In suitable embodiments, thetopoisomerase recognition site, if present, may flanked by two or morerecombination sites. In additional embodiments, the one or morerecombination sites may flank one of more restriction sites (e.g. one ormore type IIs sites) and/or the one or more selectable markers, ifpresent.

The present invention also provides vectors comprising: (a) one or moredesired nucleic acid segments; (b) optionally one or more toxic genes;and (c) one or more restriction sites (e.g. one or more type IIsrestriction enzyme recognition sites). Desired nucleic acid segmentsinclude, but are not limited to one or more genes, and one or morepromoters. Suitable restriction sites include type IIs restrictionenzyme recognition sites, such as those sites described above. Thevectors of the present invention may also comprise one or morerecombination proteins, and one or more topoisomerase recognition sitesand/or one or more topoisomerases. In suitable embodiments, thetopoisomerase recognition site, if present, may flanked by two or morerecombination sites. The vectors of the present invention optionallycomprise suitable toxic genes, as described above. The vectors of thepresent invention may also optionally include one or more selectablemarker as described throughout the specification. In another suitableembodiment, the vectors of the present invention may be “precut” by arestriction enzyme (e.g. a type IIs restriction enzyme). This precutvector may then be used to clone one more second nucleic acid moleculeswhich may comprise sticky ends compatible with the vector, oroptionally, may comprise on or more restriction sites (e.g. one or moretype IIs restriction enzyme recognition sites).

The present invention also provides methods of expressing and isolatingnucleic acid molecules and proteins comprising: (a) obtaining one ormore isolated nucleic acid molecules of the present invention; (b)introducing the isolated nucleic acid molecule into a host cell; (c)incubating the host cell under conditions sufficient to allow expressionof a nucleic acid molecule or a protein encoded by the isolated nucleicacid molecule; and (d) isolating the expressed nucleic acid molecule orexpressed protein. Host cells suitable for use in accordance with thisaspect of the invention are described elsewhere herein. Suitableincubation conditions are well known in the art and are described inFreshney, R. I., “Culture of Animal Cells: A Manual of Basic Technique,”Alan R. Liss, Inc, New York (1983) and Maniatis et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1982) and comprise incubating a host cell in a suitablegrowth medium with sufficient nutrients (e.g. Eagle's Minimum EssentialMedium, DMEM: F12 Medium, RPMI-1640 Medium, Dulbecco's Modified Eagle'sMedium, and the like) at an appropriate temperature (about 37° C.).Methods of isolation of nucleic acid molecules and expressed proteinsfrom host cells are also well known in the art and described in Manitaisid. and similar texts.

The expressed nucleic acid molecules may be suitable for use asinterfering RNA as described above. As described throughout thespecification, the expressed nucleic acid molecules will often notcomprise extraneous, undesired nucleic acids, for example nucleic acidsencoded by the one or more restriction sites (e.g. one or more type IIsrecognition sites). Similarly, the proteins produced via the methods ofthe present invention may not comprise extraneous, undesired aminoacids, for example amino acids encoded by the one or more restrictionsites (e.g. one or more type IIs recognition sites).

The present invention also provides for methods of expressing desirednucleic acid segments comprising: obtaining a product nucleic acidmolecule of the invention and incubating the nucleic acid molecule underconditions (in vitro or in vivo) such that the desired product nucleicacid molecule is transcribed and then translated. Incubation conditionsfor these methods of the invention are well known in the art as notedabove.

The present invention also provides for methods of expressing desirednucleic acid segments comprising: (a) obtaining a vector of the presentinvention; (b) introducing the vector into a host cell; and (c)incubating the host cell under conditions sufficient to allow expressionof a desired nucleic acid segment encoded by the vector. Incubationconditions for these methods of the invention are well known in the artas noted above.

Another embodiment of the present invention provides compositionscomprising the elements described above that are involved in the variouscloning methods of the invention. Such compositions comprise: (a) one ormore first nucleic acid molecules comprising one or more sticky endsthat have been generated by a restriction enzyme (e.g. one or more typeIIs restriction enzymes); (b) one or more second nucleic acid moleculescomprising one or more sticky ends which are compatible with the onemore sticky ends one the first nucleic acid molecule and, optionally,one or more selectable markers. Suitable restriction enzymes includethose described throughout the specification, including, type IIsrestriction enzyme recognition sites. The nucleic acids comprised in anyof the compositions of the present invention may optionally furthercomprise one or more selectable markers, one or more recombinationsites, one or more topoisomerase recognition sites and/or one or moretopoisomerases and described above. The compositions may comprise one ormore recombination proteins. Suitable recombination proteins include,but are not limited to, those described throughout the specification.

Another embodiment of the present invention provides compositionscomprising the elements described above that are involved in the variouscloning methods of the invention. Such compositions comprise: (a) one ormore first nucleic acid molecules comprising at least one nucleic acidsegment flanked by one or more first restriction sites (e.g. one or moretype IIs restriction enzyme recognition sites); (b) one or more secondnucleic acid molecules comprising one or more second restriction sites(e.g. one or more type IIs restriction enzyme recognition sites) andoptionally one or more selectable markers; and (c) one or morerestriction enzymes (e.g. one or more type IIs restriction enzymes) thatare specific for said first and/or second restriction enzyme recognitionsites. Suitable restriction enzymes include those described throughoutthe specification, including, type IIs restriction enzyme recognitionsites. The nucleic acids comprised in any of the compositions of thepresent invention may optionally further comprise one or more selectablemarkers, one or more recombination sites, one or more topoisomeraserecognition sites and/or one or more topoisomerases and described above.The compositions may comprise one or more recombination proteins.Suitable recombination proteins include, but are not limited to, thosedescribed throughout the specification.

The present invention also provides kits comprising the isolated nucleicacids and/or vectors of the present invention. These kits are useful forpracticing the various methods of the invention. Kits may comprise oneor more first nucleic acid molecules and one or more second nucleic acidmolecules. The first nucleic acid molecule may be an isolated nucleicacid molecule of the invention and the second nucleic acid molecule maybe a vector of the present invention.

Kits of the invention may contain any number of components but typicallywill contain at least two components. Kits according to this aspect ofthe invention may comprise one or more containers, which may contain oneor more components selected from the group consisting of one or morenucleic acid molecules or vectors of the invention, one or more primers,one or more polymerases, one or more reverse transcriptases, one or morerecombination proteins, one or more restriction enzymes (e.g. one ormore type IIs restriction enzymes, or other enzymes for carrying out themethods of the invention), one or more topoisomerases, one or morebuffers, one or more detergents, one or more restriction endonucleases,one or more nucleotides, one or more terminating agents (e.g., ddNTPs),one or more transfection reagents, pyrophosphatase, and the like. Thekits of the invention may also comprise instructions for carrying outmethods of the invention.

It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein may be made without departing from thescope of the invention or any embodiment thereof. Having now describedthe present invention in detail, the same will be more clearlyunderstood by reference to the following examples, which are includedherewith for purposes of illustration only and are not intended to belimiting of the invention.

EXAMPLES Example 1 Expression of Interfering RNA Using a SeamlessCloning Vector

The expression of short interfering hairpin RNA molecules (shRNA) invivo can decrease the expression of genes with complementary sequencesby RNA interference (RNAi) as described previously. The seamless cloningvector described here (pENTR/U6) allows for rapid and efficient cloningof double-stranded oligonucleotide pairs (˜47 bp) coding for a desiredshRNA target sequence into a Pol III U6 expression cassette. Theresulting shRNA vector contains an RNAi cassette flanked by attL sites.Therefore, the pENTR/U6 shRNA vectors can be used directly for transienttransfection to test various shRNA target sequences, as well as totransfer the best shRNA cassettes to Lenti and Adenoviral DEST vectorsfor delivery into “hard to transfect” cells.

Kit Components.

Purified, BsaI-linearized pENTR/U6.2 (once it is cut with BsaI, i.e. thelinear vector is called pENTR/U6) (Catalog No. K4945-00 and K4944-00,Invitrogen, Corp., Carlsbad, Calif.) Annealed lamin A/C control oligos:Top 5′-CACCGTGTTCTTCTGGAAGTCCAGCGAACTGGACTTCCAGAAGA ACA (SEQ ID NO:9),Bottom 5′-AAAATGTTCTTCTGGAAGTCCAGTTCGCTGGACTTCCAGAAGAACA C (SEQ IDNO:10), Sequencing primers: U6 forward 5′-GGACTATCATATGCTTACCG (SEQ IDNO:11), M13 reverse 5′-CAGGAAACAGCTATGAC (SEQ ID NO:12) (Catalog No.N530-2, Invitrogen, Corp., Carlsbad, Calif.), T4 DNA ligase (Catalog No.15224-025, Invitrogen, Corp., Carlsbad, Calif.) 5× T4 DNA ligase buffer(Catalog No. Y90001, Invitrogen, Corp., Carlsbad, Calif.), OneShot Top10cells (Catalog No. C4040-03, Invitrogen, Corp., Carlsbad, Calif.). Thus,exemplary kits of the invention may comprise one, more, or all of thesecomponents.

Vector Construction.

Entry vector. The nucleic acid sequence of pENTR U6.2 (BsaI-ccdB) isshown in Table 5, SEQ. ID. NO:1. The U6 promoter sequence was PCRamplified from genomic DNA (primers: 5′-AAGGTCGGG CAGGAAGAGGG-3′ (SEQ IDNO:13); 5′-AGCGAGCACGGTGTTTCGTC-3′ (SEQ ID NO:14)) and TOPO cloned intopCR2.1/TOPO (included in kits, Catalog Nos. K4500-01, K4500-40,K4550-01, K4550-40, K4560-01, K4560-40, K4520-01 and K4520-40,Invitrogen, Corp., Carlsbad, Calif.). The promoter sequence wassubsequently PCR amplified with the same primer sequences but withAsp718 and NotI sites appended to the primer 5′ ends(5′GTGGGTACCAAGGTCGGGCAGGAAG AGGG-3′ (SEQ ID NO:15;5′-GTGGCGGCCGCGGTGTTTCGTCCTTTCCACAAG-3′ (SEQ ID NO:16)). This PCRproduct was cloned by Asp718-NotI sticky end ligation into an Entryvector with the pENTR/1a polylinker (Catalog No. 11813, Invitrogen,Corp., Carlsbad, Calif.) and pDONR/221 backbone (Catalog No. 12536-017,and provided in kits 12537-023, 12538-013, 12535-019, Invitrogen, Corp.,Carlsbad, Calif.). The ccdB gene was amplified from pLenti6/V5/DEST(Catalog Nos. V496-10 and K4960-00, Invitrogen, Corp., Carlsbad, Calif.)(primers: 5′-GTGGCGGCCGCAAAGATCCTCCAGTGGATCCGGCTTAC TAAAAG-3′ (SEQ IDNO:17); 5′GTGCTCGAGAAAAAAGTCGACACGGAGCCCTCC AGTTATATTCCCCAGAACATCAGG-3′(SEQ ID NO:18)) and cloned into the above vector at the NotI and XhoIsites. These primers introduced BpmI restriction enzyme sites in theproper position at the ends of the PCR product and a 6 bp polyT Pol IIIterminator.

To engineer the BsaI vector, a double stranded oligo containing a BsaIsite and NotI site (5′GAGACCGCGGCCGCTTCTCGAGGTCTCATT (SEQ IDNO:19)+5′TGAGACCTCGA GAAGCGGCCGCGGTCTCCG-3′ (SEQ ID NO:20)) was clonedinto BpmI-digested plasmid. The resulting plasmid was digested with NotIand XbaI and ligated to a new ccdB region PCR amplified (primers:5′CACGCGGCCGCTGGATCCGGCTTACTAAAAG-3′ (SEQ ID NO:21); 5′CACTCTAGAAAAAATGAGACCTTATATTCCCCAGAACATCAGG-3′ (SEQ ID NO:22)) with a NotI site onone end and a BsaI site, 6 bp polyT Pol III terminator, and XbaI site atthe other. The final construct is named pENTR/U6.2 (BsaI-ccdB).

LacZ expression control vector. The LacZ expression control plasmid,pcDNA2.2 MS/GW/LacZ was made using Multi-site Gateway (CMVlacZV5).pENTR5′-CMV, pENTR-LacZ and pENTR/V5TKpolyA were mixed with the DESTR4R3 plasmid using LR Plus Clonase. The three plasmids in the Multi-sitereaction were all created by a standard Gateway recombinationreaction: 1) the CMV promoter was amplified from pcDNA3.1 (Catalog No.V790-20 and V795-20, Invitrogen, Corp., Carlsbad, Calif.) using primersflanked with attB4 and attB1 sequences and recombined with pDonr5′(P4-P1R) to form pENTR5″-CMV. 2) The LacZ gene was amplified frompcDNA3.1-LacZ using attB1 and attB2 flanking primers and recombined withpDonr 221 to create pENTR-LacZ, and, 3) the V5-TKpolyA element wasamplified from pcDNA3.2 using attB2 and attB3 primers and recombinedwith pDonr3′(P2-P3R).

Preparation of linear pENTR/U6.2 ready for cloning. pENTR/U6.2 in DB3.1cells was grown in LB media with 50 μg/ml kanamycin. Plasmid DNA waspurified by SNAP midi prep with a yield of 67 μg/50 ml of culture. Tenμg of vector was digested with BsaI at 50° C. in 200 μl with 5 units ofBsaI/μg of DNA for 2 hrs. After addition of 1.5 vol of SNAP miniprepbinding buffer, the reaction was added to a SNAP miniprep column, washedaccording to the SNAP protocol for miniprep DNA, and eluted in 100 μlddH2O and stored at −20° C.

ShRNA Oligonucleotide Annealing. DNA oligonucleotides of 46-53 nt wereproduced with desalt purification only. Individual oligos were dilutedin ddH2O to a final concentration of 200 μM as verified byspectrophotometric analysis at OD₂₆₀. Complementary oligos were mixed tothe final desired concentration with either: 1) TE (10 mM Tris pH 8.0, 1mM EDTA), 2) 10× Annealing Buffer and ddH2O such that the final, 1×buffer was 10 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, or 3) the samebuffer as in 2 but with a final concentration of 10 mM MgCl₂. (Forexample, to create a 50 μM stock of a ds-oligo in 20 μl, 5 μl of each200 μM ss complementary oligo was mixed with 2 μl of 10× Annealingbuffer and 8 μl of ddH2O). Mixed oligo pairs were heated and cooled ineither an MJ thermocycler (94° C. for 2 min, then decreased by 0.1° C.every second to 25° C., and stored at 4° C.) or incubation in a 95° C.bath for 4 min, then cooling to room temperature over 15 min beforeputting the sample on ice. Annealed ds-oligos were diluted to thedesired concentration with TE at room temperature.

Cloning target site DNA oligos into pENTR/U6. BsaI cut pENTR/U6.2 andds-oligos were incubated in a 20 μl reaction using 5 times ligase bufferand 1 μl ligase for 5 min at room temperature. Two microliters of theligation reaction were added to chemically competent Top10 One Shotcells (Catalog Nos. C4040-10, C4040-03, C4040-06, Invitrogen, Corp.,Carlsbad, Calif., ˜50 μl), incubated on ice for 20 min, heat shocked at42° C. for 30 sec., and placed back on ice, followed by the addition of250 μl SOC and incubation at 37° C. (shaking) for 1 hr. Ten to onehundred microliters of this transformation reaction were plated on LBKan (50 μg/ml) agarose plates.

The number of colonies per plate was determined after an overnightincubation at 37° C. A supercoiled pUC19 (2 μl of a 10 pg/μl stock)transformation control was performed with each set of cells transformed;in this case the transformation efficiency is reported as number ofcolony forming units per microgram.

Sequence analysis of pENTR/U6 shRNA target clones. Plasmid DNA wasisolated from pENTR/U6 clones using the SNAP mini prep kit (Catalog No.K1900-01, Invitrogen, Corp., Carlsbad, Calif.) under standardconditions. Two different primers were used for sequence analysis:

-   -   1) U6 forward, 5′-GGACTATCATATGCTTACCG (forward primer, binds in        U6 promoter 55 bp from the 3′ end of the U6 promoter) (SEQ ID        NO:11)    -   2) M13 R, 5′-CAGGAAACAGCTATGC (reverse primer, binds        “downstream” from the AttL2 site, 146 bp from the pol III        termination) (SEQ ID NO:12)

Gateway L×R recombination. 150 ng of each pENTR/U6 shRNA clone and 150ng of pLenti6/PL-DEST or 300 ng of pAD/PL-DEST (FIG. 10) (Catalog No.V494-20, Invitrogen, Corp., Carlsbad, Calif.) were incubated in a 20 μlreaction using the 5× buffer and 5×LR Clonase enzyme mix, and incubatedat 25° C. for 1 hr. Two microliters of this L×R reaction weretransformed into chemically competent cells as described above exceptthat selection plates had 50 ug/ml ampicillin instead of kanamycin.

ShRNA Transfections.

All transfections were carried out in 24-well plates. For luciferase andβ-galactosidase (β-gal) knockdown experiments, 600 ng of pENTR/U6-shRNAvectors were cotransfected with 100 ng each pcDNA5/FRT/luc and thepcDNA1.2/V5-GW/lacZ positive control plasmid into GripTite™ 293 cells(Catalog No. R795-07, Invitrogen, Corp., Carlsbad, Calif.) usingLipofectamine 2000™. Briefly, cells were plated the day beforetransfection in 0.5 ml medium lacking antibiotics at 2×10⁵ cells perwell. On the day of transfection, cells were typically 90-95% confluent.For each well, 2 μl of Lipofectamine 2000™ were diluted with 48 μlOptiMEM, incubated 5 min at room temperature, then mixed with DNAsdiluted with OptiMEM to 50 μl. Complexes were incubated an additional 20min at room temperature before addition to cells. Medium was changed 3hr after transfection to minimize toxicity.

Luciferase and β-Gal Assays.

After 48 hr, GripTite™ 293 cells were lysed in 0.5 ml luciferase lysisbuffer (25 mM Tris-HCl pH 8.0, 0.1 mM EDTA pH 8.0, 10% glycerol, 0.1%Triton X-100) and subjected to a −80° C. freeze-thaw. 50 μl of eachlysate was used in a luciferase luminescence assay (Promega) whileanother 10 ul was used in a β-gal luminescence assay (Tropix) accordingto the manufacturers' instructions.

Results

The vector pENTR/U6 is designed to express shRNA in mammalian cells foruse in RNAi. (pENTR/U6.2 is the supercoiled vector containing the ccdBgene; once linearized with BsaI, the vector will be referred to aspENTR/U6.) pENTR/U6 allows the cloning of shRNA target sequences betweenthe human U6 pol III promoter and a 6 T termination signal in a GatewayEntry (ENTR) vector. In this case, the entire RNAi cassette (U6promoter, cloning site, and termination signals) is between the attL1and attL2 recombination sites. Therefore, U6 driven expression of anshRNA is possible directly from ENTR vector and does not requiresubsequent L×R transfer to a DEST vector.

Vector Preparation.

pENTR/U6.2 (BsaI-ccdB) is digested with the type IIS restriction enzymeBsaI in preparation for cloning ds-oligos (˜47 mers) containing shRNAtarget sequences. Type IIs restriction enzymes cut outside of theirrecognition sequence and can therefore be used to create sticky ends ofany sequence in the vector. In this case, the BsaI digest leaves the 4nt 5′ ssDNA end 3′-GTGG-5′ at the end of the U6 promoter and the singlestranded 3′-TTTT-5′ at the other vector end (the first four Ts of thetermination signal).

Digestion of the pENTR/U6.2 by BsaI generates three fragments (2850,577, and 91 bp). The linearized cloning vector is 2850 bp; smallerfragments derive from the ccdB gene (ccdB has a BsaI site). Removal ofthe smaller fragments from the final vector prep is not required;however, the amount of the 91 bp fragment recovered from the SNAPpurification can vary. Uncut pENTR/U6.2 or clones that have reassembledthe functional ccdB gene will not propagate in Top10 cells. The cloningefficiency of either small fragment alone is very low due tonon-compatible ends.

Insert Annealing

A five-minute bench top ligation and subsequent transformation is highlyefficient at cloning dsDNA oligo shRNA target sequences—if the oligoinserts are properly annealed. A typical 46 nt ss-oligo is made of a 4nt 5′ cloning overhang followed by 19 nt of “sense” and a complementary19 nt “antisense” sequence connected by short 4 nt “loop.” Thus theoligos can form a ˜19 bp DNA intra-molecular hairpin. Therefore,conditions must be optimized to favor intermolecular annealing betweentwo different complementary oligos rather than the production ofsingle-strand intramolecular hairpins. The formation of intermoleculards-oligos can be accomplished by melting (heating to 94° C.) and coolingcomplementary oligos at high concentrations in the appropriate buffer.

Intermolecular double-stranded molecules can be formed in annealingbuffers containing either 20 or 100 mM NaCl when the oligo concentrationis 50 μM during the heating and cooling cycle. The ds-molecules can beseparated from the single-stranded hairpins in an E-gel. Additionally,no difference was noted between using the Thermocycler or water bathprotocols to melt/cool the reaction.

Upon closer examination of the salt and oligo concentration, a bufferwithout any NaCl (TE) would not support formation of ds-47mers even at100 μM concentrations, adding MgCl₂ to 100 mM NaCl had no effect, andoligo concentrations of less the 50 μM were compromised in the amount ofds-47mers created.

Once created, the dsDNA 47 mer shRNA inserts can be diluted in TE forcloning. After the ds-47 mers are diluted, they are stable at 4° C.overnight, but will form single strand hairpins if melted, i.e.incubated at temps above 42° C.

Heating and cooling of shRNA target oligos at concentrations of 50 μM orgreater in 10 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA creates a mixtureof ˜50:50 ds/hairpin molecules which can be effectively cloned into BsaIlinearized pENTR/U6 (see pENTR/U6 cloning, below).

Gateway ENTR Vector Testing.

The supercoiled pENTR/U6.2 (BsaI-ccdB) vector, prior to linearizationfor cloning, passes the criteria set for Gateway ENTR vectors (>104killing by ccdB). Supercoiled pENTR/U6.2 was transformed into E. colicells it should kill (Top10 and HB101 cells) as well as the DB3.1 cellline designed to propagate plasmids with the ccdB gene. pENTR/U6.2transforms DB3.1 cells 1.3×10⁴ times better than Top10s cells once thenumber of colonies per plate are adjusted for the differenttransformation efficiencies of the different cell lines (the Top10 cellswere ˜200 times more competent than the DB3.1 cells and ˜400 times morecompetent than the HB101 cells).

When BsaI digestion of pENTR/U6.2 is complete, most of the supercoiledvector is linearized. Transformation of BsaI cut, SNAP purified pENTR/U6vector only generated a small number of “background” colonies per platein Top10 or DB3.1 cells. Eight colonies were obtained in DB3.1 cells andall looked like the parent sc pENTR/U6.2 by RFLP analysis (data notshown) indicating the BsaI digest is efficient and only a small fractionof the plasmids are left uncut after the 2 hr incubation. In Top10 cellsonly 4 colonies were obtained; RFLP analysis of these indicated twoclasses, neither of which was the parent plasmid (possibly pENTR/U6closed without the ccdB gene and one fragment of the ccdB genere-cloned).

pENTR-U6 Cloning.

A five-minute bench-top ligation is an easy and efficient method toclone shRNA target sequences into pENTR/U6. The cloning process wasoptimized over a wide range of vector concentrations (20 pg-5 ng) andinsert concentrations (0.4 pg-10 ng) with the shRNA target sequencelacZ-19. All the optimization of the cloning reaction was done withds-oligos annealed at a concentration of 50 μM prior to dilution in TEand transformation into chemically competent Top10 cells. Sequenceanalysis of the shRNA clones demonstrate that >90% have inserts in thecorrect orientation.

Greater than 15 other ds-oligo inserts, each with a different shRNAtarget sequence, have been cloned into pENTR/U6 under comparableconditions. In all cases, the number of colonies generated was similarto the numbers of colonies generated with the lacZ-19 ds-oligo. Nosignificant difference has been noted in how different inserts cloneinto the pENTR/U6 vector.

Sequence Analysis.

The efficiency of cloning shRNA target-sequence inserts was determinedby sequence analysis through shRNA target sequences. Analysis of thelacZ-19 shRNA target inserts cloned in pENTR/U6 under the recommendedconditions, demonstrated that 100% (38/38) of the randomly selectedclones have an insert cloned in the correct orientation.

Sequence analysis with the U6 forward primer provides excellent sequencethrough the cloned shRNA target sequence. It is designed for ease ofanalysis of the cloned oligos, binds the U6 promoter inside the attLsites 55 bases from the cloning junction, and allows for the analysis ofthe entire cloned insert with a 100 base “read” before the “downstream”attL2 site.

RNAi by Transient Transfections.

Post-transcriptional inhibition of luciferase (GL2) and lacZ expressionwas evident upon expression of shRNA targets from the pENTR/U6 vector(FIG. 3A). Specific inhibition is evident with pENTR/U6 shRNA clonestargeting Luciferase and lacZ expression from co-transfected reporterconstructs. The Luciferase pENTR/U6 GL2-22 construct inhibits expressionof GL2 Luciferase but not lacZ (FIG. 3A); similarly, the pENTR/U6 withthe lacZ-19 shRNA target sequence (the target provided as a control inthis kit) inhibits lacZ expression from pcDNA1.21V5-GW/lacZ (the controlexpression vector for this kit)—but not Luciferase (FIG. 3B).

Similar inhibition of both lacZ and Luciferase is evident with shRNAsthat target different sites, although not all shRNA sequences areeffective (FIGS. 4A and 4B). The kit control lacZ-19 target sitepresented in FIG. 4B is the same shRNA target site used in FIG. 3B, andonly the lacZ4-AS sequence inhibits expression to the same degree. ThelacZ4-SA only moderately inhibits expression and the lacZ5 clones havelittle if any inhibitory effect. Similarly, the GL2sh2 and GL2-22 (AS)target sites are the most effective shRNA clones tested at inhibitingluciferase expression (FIGS. 4A and 4B). Interestingly, the sense toanti-sense orientation of the shRNA target sequence can make aconsiderable difference in the level of inhibition at a specific target(FIGS. 4A and 4B). However, the optimal orientation(sense-loop-antisense (SA) or antisense-loop-sense (AS)) is not clear;with Luciferase, the AS orientation was most effective, but with lacZthe SA orientation was most effective (FIG. 4A, ENTR/U6-A6-GL2-22 AS vs.SA, and FIG. 4B, ENTR/U6-A6-lacZ4-AS vs. SA).

Additionally, the lacZ-19 shRNA target sequence was tested inderivatives of the pENTR/U6 vector with terminators of 4-8 Ts. All theterminators behaved similarly (FIG. 5).

Gateway L×R Cross.

Any shRNA target sequence cloned into pENTR/U6 can easily be transferredas a U6 RNAi cassette to a Gateway DEST vector by attL×attR (L×R)recombination at the att sites. Following is a demonstration of theefficiency of L×R transfer. The lacZ-19 target sequence cloned intopENTR/U6 was transferred into pLenti6/PL-DEST and pAD/PL-DEST by astandard L×R Clonase catalyzed recombination reaction (See, e.g., FIGS.38 and 39) as described previously (See U.S. Pat. Nos. 5,888,732;6,143,577; 6,171,861; 6,277,608; and 6,720,140; the disclosures of whichare incorporated by reference herein in their entireties). Additionally,12 different pENTR/U6 shRNA target subclones, including target sequencesto Lamin AC and Luciferase, were also recombined into these two DESTvectors. In all cases, the L×R crosses were efficient. When 2 μl/20 μlL×R reaction were transformed and ⅙th (50 μl of the transformationreaction plated, 300-800 colonies/plate were obtained in Top10 cells.Even in HB101 cells that were ˜40 fold less competent to take up DNAthan the Top 10 cells, 10-20 colonies/plate could be obtained by platingmore of the transformation reaction (100 μl vs. 50 μl). Note that thenumber of clones obtained are similar between the Lenti DEST and theAdeno DEST vectors, even though the Adenoviral vector is almost 4 timesthe size of the Lentiviral vector (˜36 kb vs. ˜8.6 kb).

The L×R crosses were not only efficient but also effective. Ten out often of the Adeno DEST vector recombinants had the correct RNAi cassetteas determined by RFLP analysis. pLenti DEST recombinants weretransformed into both Top10 and HB101 E. coli cells because HB101 cellsare known for reducing the recombination between the lentiviral LTRsequences. In this case, 10/10 recombinants were correct using HB101cells.

shRNA Target Site Selection

The present invention may be used to create shRNAs with any desired stemlength, orientation, and loop sequence. In general, target sequencesshould be complex (no runs of more than 3 of the same nucleotide), withlow GC content (30-50%), and avoid known RNA-protein interaction sites.Target sites should be a minimum of 19 nt, and sites of up to 29 nt areeffective.

DNA Oligo Insert Design

Once a candidate target site has been selected, it must be convertedinto an shRNA sequence, and the DNA oligos ordered for cloning intopENTR/U6. The shRNA sequence can be in two possible orientations. Eitherthe sense target site or the antisense sequence of the target site canbegin the shRNA, followed by a short loop sequence and then the oppositestrand of the target site.

The fact that the polymerase (pol III) will terminate transcriptionafter 4 thymidines (Ts) constrains the oligo design. Strings of morethan 3 Ts should be avoided in the middle of a target site, or with anyTs in the connecting “loop”, to prevent early termination. Additionally,Ts at the 3′ end of the target will abut the polyT terminator and maycause slightly premature termination. Changing the sense/antisenseorientation of the shRNA may be necessary for specific target sites toavoid early pol III termination by positioning different sequences nextto the loop or polyT terminator.

Additionally, the native U6 snRNA initiates at a guanosine (G), and this+1 base is believed to be important. Although this system allowsadvanced users to choose any +1 base, we have designed all of ourinserts to initiate at a G. In cases where the G is part of the targetsequence, it is simply incorporated into the stem, with a complementarycytosine base placed just before the terminator. When G is not the firstbase in the sense or antisense target sequence, it is added to the 5′end of the shRNA with no complementary base at the 3′ end. If use of a Gis not desired, an A is believed to be better than an C or T.

Functional loops of anywhere from 4 to lint have been reported in theliterature. Short loops are preferred as they reduce the lengths of theoligos needed for cloning. 5′-TTCG, 5′-AACG, and 5′CGAA have been usedas the loop sequences in successful RNAi constructs. However, loopscontaining thymidines must be avoided in some cases as they may causeearly termination, as discussed above.

Finally, to convert an shRNA sequence into an oligo pair for insertion,5′CACC-3′ was added to the 5′ end of the shRNA sequence to create the“top” oligo. The “bottom” oligo is the complimentary sequence of the topoligo with the 5′CACC-3′ removed and 5′AAAA-3′ appended to the 5′ end.

Conclusion

The pENTR/U6 and Gateway DEST vectors are the cornerstones of a superiorsystem to clone shRNA target sequences into an RNAi expression cassetteand deliver it to cells (FIG. 28). Two other commercial sources withsimilar pol III vectors (Ambion with pSilencer, and OligoEngines withpSuper) require the synthesis of longer insert oligos (˜70 nt and 55 ntrespectively) because their cloning schemes need the end of the U6promoter and termination signals to be “built-back” with the insert.Additionally, their cloning protocols call for ligation incubations of 1hr or greater compared to the 5 min bench-top reaction described here.This is likely due to the PEG present in the present ligation buffer, aswell as the present vector design features that eliminate background(the ccdB negative selection and the non-compatible ends left after BsaIdigestion). The present invention also has the Gateway Advantage; anyinsert cloned and sequence verified in pENTR/U6 is then available forany application made possible by the DEST vectors—such as viral deliveryof shRNA by Virapower™.

The demonstrations of RNAi in transient transfections reported here, aswell as examples of successful RNAi by transduction indicate the U6promoter can generate sufficient shRNA for RNAi. Experiments that definethe rules required for efficient RNAi will make this vector all the morevaluable.

Example 2 Expression of Interfering RNA Using a Seamless Cloning VectorAbstract and Introduction

Short hairpin RNA (shRNA) expression cassettes built into the U6 RNAiEntry Vector can be used to transiently knockdown genes of interest incell culture. However, the Entry Vector carries no marker for selectionin mammalian cells, and the plasmids must be introduced into cells bytransfection. Transfection efficiency varies widely between cell linesand is ineffective in primary and terminally differentiated cells. Incontrast to plasmid transfection, lentiviral delivery allows simple,stable transduction of a wide variety of cell types including primaryand terminally differentiated cells. A number of recent publicationsdescribe the use of lentiviruses to deliver shRNAs to mammalian cells(Abbas-Terki et al. 2002, Dirac & Bernards 2003, Matta et al. 2003, Qinet al 2003, Rubinson et al 2003, Stewart et al 2003, Tiscornia et al.2003), demonstrating an existing interest in this technique.

Invitrogen offers several Gateway-adapted lentiviral vectors for cloningof coding sequences downstream of a Pol II promoter. However, thepresence of such an upstream promoter may interfere with Pol IIIexpression from a U6 cassette. A promoterless Destination vector,pLenti6/RNAi-DEST has been created with attR1 and attR2 sites compatiblewith the U6 RNAi Entry Vector. A map of pLenti6/RNAi-DEST is shown inFIG. 6A. pLenti6/RNAi-DEST allows simple and reliable transfer of shRNAexpression cassettes into the lentiviral backbone. The viral vectorconfers blasticidin resistance for selection of stably transduced cells.Transduction by lentiviruses expressing lamin A/C shRNAs is demonstratedto efficiently and specifically knock down endogenous protein levels.pLenti6/RNAi-DEST complements the ViraPower™ product line and provides apowerful new application for the U6 RNAi Entry Vector.

Key Performance Criteria for Lenti6/RNAi-DEST include: (1)pLenti6/RNAi-DEST passing standard manufacturing QC specs forDestination vectors. (2) Gateway cloning shRNAs into pLenti6/RNAi-DESTand packaging virus at levels comparable with regular vectors. (3)Showing specific knockdown of endogenous lamin A/C gene.

Materials and Methods

Construction of pLenti6/RNAi-DEST Vector pLenti6/RNAi-DEST is theproduct of a Gateway B×P reaction between pLenti6/PL/attB4/V5/GW-GFP andpDONR 221. The B×P reaction was transformed into DB3.1 and selected onLB media containing Ampicillin (100 μg/ml) and chloramphenicol (15μg/ml). Colonies of the transformants were analyzed by restrictiondigest. A map of pLenti6/RNAi DEST is shown in FIG. 6A.

ShRNA-Containing Entry Clones

The various shRNA-containing Entry clones used are set out in Table 1.The hairpins are targeted to sites on the lamin A/C or luciferase genesas indicated. All entry clones were created by oligo cloning intopENTR/U6.2. Loops and stems choices are described in Example 1.

TABLE 1 pENTR/U6 Entry Clones Loop Stem length^(b) Target Clone nameTarget gene Orientation^(a) sequence (bp) position^(c) (nt) pENTR/U6-lamin A/C SA UUCG 19 610-628 lamAC-SA-uucg pENTR/U6- lamin A/C AS UUCG19 610-628 lamAC-AS-uucg pENTR/U6- lamin A/C AS CGAA 19 610-628lamAC-AS-cgaa pENTR/U6- lamin A/C SA CGAA 19 610-628 lamAC-SA-cgaapENTR/U6-GL2- luciferase AS UUCG 22 153-174 22 pENTR/U6- luciferase ASGAACGT 29 1355-1383 GL2sh2^(d) TG ^(a)Orientations are eithersense-loop-antisense (SA) or antisense-loop-sense (AS). ^(b)Stem lengthdoes not include +1 G base if it is not also part of the target site.^(c)Target position is relative to start codon. ^(d)Hairpin design basedon a previously assessed technology from Cold Spring HarborLaboratories.

Destination Vector QC and Generation of Expression Control Vector

pLenti6/RNAi-DEST was monitored for quality using the official “DestVector QC Procedure” established by manufacturing. The expressioncontrol plasmid, pLenti6/RNAi/U6-GW/lamAC was generated by a standardGateway L×R reaction between pLenti6/RNAi-DEST andpENTR/U6-lamAC-AS-cgaa. Clones of pLenti6/RNAi/U6-GW/lamAC wereconfirmed by restriction analyses. A map of pLenti6/RNAi/U6-GW/lamAC isshown in FIG. 6B.

Cell Culture

293FT cells were cultured in DMEM/10% FBS/L-glutamine/non-essentialamino acids/penicillin/streptomycin containing 500 μg/ml G418. HeLacells were cultured in DMEM/10% FBS/L-glutamine/non-essential aminoacids/penicillin/streptomycin.

Virus Production

For virus production, 1×10⁷ 293FT cells were plated per T175 flask.Twenty-four hours later, culture medium was replaced with 20 mlOptiMem/10% FBS, and shRNA-encoding viruses were packaged byco-transfecting the 293FT cells with the respective lentiviral vectorand pLP1, pLP2 and pLP/VSVG (at a mass ratio of 1:1:1:1, 24 μg of totalDNA) as follows: The 24 μg DNA was mixed with 3 ml of OptiMem media. Ina separate tube, 72 μl of Lipofectamine 2000 was also mixed with 3 ml ofOptiMem media. After a 5-minute incubation period at room temperature,the two mixtures were combined and incubated at room temperature for anadditional 20 minutes. At the completion of the incubation period, thetransfection mixture was added to the cells dropwise and the flask wasgently rocked to mix. The following day the transfection complex wasreplaced with 30 ml complete media (DMEM, 10% FBS, 1%penicillin/streptomycin, L-glutamine and non-essential amino acids).Virus-containing media were harvested at day 2 and day 3post-transfection, centrifuged at 3000 rpm for 5 minutes to remove deadcells, and filtered through sterile 0.45 micron cellulose acetatefilters to remove fine debris. Viruses in the filtrates wereconcentrated by ultracentrifugation (90 minutes, 23000×g, 4° C.). Viralpellets from ultra-centrifugation were resuspended in 500-600 μl growthmedia. One hundred-microliter aliquots of concentrated virus were storedin −80° C. freezer until use.

Viral Titering and Transduction

All applications of virus to cells were performed in the presence of 6μg/ml polybrene (Sigma, hexadimethrin bromide, #H9268) and media changeswere performed 12-24 hours post transduction. For titering virus, 6-wellplates were seeded with 2×10⁵ HT1080 cells per well the day beforetransduction. One milliliter each of ten-fold serial dilutions of viralsupernatant ranging from 10⁻² to 10⁻⁸ was prepared. All dilutions weremixed by gentle inversion prior to adding to cells. Mock-transducedcells had no virus added to them. Plates were gently swirled to mix. Thefollowing day, the media was replaced with complete media. Forty-eighthours post-transduction, the cells were placed under 10 μg/mlblasticidin selection. After 7 to 10 days of blasticidin selection theresulting colonies were stained with crystal violet: A 1% crystal violetsolution was prepared in 10% ethanol. Each well was washed with 2 ml PBSfollowed by 1 ml of crystal violet solution for 10 minutes at roomtemperature. Excess stain was removed by two 2 ml PBS washes andcolonies visible to the naked eye were counted to determine the viraltiter of the original supernatants.

Transductions to test shRNA activities were performed in the appropriatecells in 12-well plates. Cells were plated at 1×10⁵/well twenty-fourhours before transduction. The next day, the media was replaced withcomplete media. Transduction was conducted in a final volume of 500 μland contained the appropriate volumes of virus supernatant to achieve arange of MOIs.

Cell Lysis and Western Blot

Cell lysis for lamin A/C and beta-actin western blots were performed asfollows: Forty-eight or 120 hours post-transduction, cells wereharvested with Versene (Invitrogen), transferred to microfuge tubes, andcentrifuged at 3000 RPM for 4 min. Pellets were lysed in 2× NuPAGE® LDSSample Buffer with 1× Sample Reducing Agent and denatured at 95° C. for5 min prior to electrophoresis. Protein samples were electrophoresed onNuPAGE® Novex 4-12% Tris-Bis Gels in 1×MOPS-SDS buffer with NuPAGE®Antioxidant in the upper chamber. Western blot analyses were performedusing the Western Breeze Immunodetection Kit (Invitrogen) according tothe manufacturer's protocol. Lamin A/C and beta-actin proteins weredetected using 1:1000 monoclonal anti-lamin A/C (BD Biosciences) and1:5000 monoclonal anti-beta-actin (Abcam) antibodies, respectively.

Results and Discussion

Destination Vector QC pLenti6/RNAi-DEST passed the standardmanufacturing QC specs for Destination vectors with respect to totalcolony count (Table 2) and ccdB assay (Table 3).

Virus Titers

ShRNA-encoding lentiviral vectors were used to produce virus in 293FTcells. The vectors produced viral titers comparable to titers attainedwith regular lentiviral vectors that do not contain shRNA (Table 4).This indicated that introduction of shRNAs into the lentiviral backbonedoes not compromise virus packaging or transduction efficiency.

TABLE 4 Lenti6/RNAi Virus Titers Crude Concentrated Virus Titer VirusTiter Virus (cfu/ml) (cfu/ml)^(a) Lenti6/RNAi/U6-GW/ 1.00E+6 4.30E+08lamAC-SA-uucg Lenti6/RNAi/U6.2-GW/ 2.10E+6 5.85E+08 lamAC-AS-uucgLenti6/RNAi/U6.2-GW/ 8.00E+5 1.35E+08 amAC-AS-cgaa Lenti6/RNAi/U6.2-GW/1.20E+6 4.45E+08 lamAC-SA-cgaa Lenti6/RNAi/U6-GW/GL2-22 6.00E+5 4.50E+08Lenti6RNAi/U6-GW/GL2sh2 1.30E+6 5.20E+08 Lenti6/V5-GW/GFP 4.00E+5 8.0E+07 (non-RNAi virus) ^(a)Concentrated from two 175 cm² flasks each.

Knockdown of Lamin A/C

Lentiviruses were tested for their ability to deliver shRNAs tospecifically knock down lamin A/C expression in HeLa cells. Lentivirusesexpressing luciferase-targeted shRNAs served as negative controls.Inhibition of lamin A/C expression was analyzed by western blot. ShRNAstargeted to lamin inhibited expression of both lamin A and C isoforms 48hr and 5 days post-transduction (FIG. 7). The extent of inhibitiondepended on transduced MOI, indicating knockdown was dose-dependent.Lentiviruses encoding shRNAs lamAC-AS-cgaa and lamAC-SA-cgaa providedthe best lamin knockdowns (FIG. 7, top panel lanes 11-16; bottom panellanes 14-19). Of the two shRNAs, lamAC-AS-cgaa mediated robustinhibition even at the relatively low MOI of 14 (FIG. 7, top panel lane11 and bottom panel lane 14). The lamin A/C shRNAs had no effect onbeta-actin expression irrespective of transduced MOI (FIG. 7, beta-actinblots). Control luciferase shRNAs had no effect on beta-actin expression(FIG. 7, top panel lanes 7-9 and 17-19; bottom panel lanes 1-3 and11-13) and minor effect on lamin A/C expression even at the very highMOI of 520 (FIG. 7, top panel lane 19; bottom panel lane 13). Theseresults show specific inhibition of lamin expression with lamin-targetedshRNAs. The inhibition is not the effect of general inhibition of geneexpression. Results of the control shRNA transduction provide furtherevidence of the specific activity of the lamin-directed shRNAs.

pLenti6/RNAi has also been used to specifically knock down luciferase(75% inhibition, 48 hrs post-transduction in Flp-In 293 luc cell line;data not shown) and lacZ at high MOIs (55% inhibition, 96 hrspost-transduction in HT1080LacZ cells; data not shown). These providefurther evidence that pLenti6/RNAi-DEST vector will function with otherRNAi cassettes.

Summary

Gateway-adapted lentiviral vector pLenti6/RNAi-DEST has been developedfor RNAi analyses. pLenti6/RNAi-DEST is designed to be used in L×Rreactions with pENTR/U6. pLenti6/RNAi-DEST meets the performancecriteria for all DEST vectors as well as criteria for packaging andtransducing lentiviruses. Viruses Lenti6/RNAi/U6-GW/lamAC-AS-cgaa andLenti6/RNAi/U6-GW/lamAC-SA-cgaa transduce shRNAs that specifically knockdown lamin A/C expression. The lamAC-AS-cgaa hairpin was chosen as thepositive control for the U6 RNAi Entry and pLenti6/RNAi Kits. Thesequence of lamAC-AS-cgaa hairpin is shown in the Kit Components andConfiguration below.

Example 3 RNAi Using Block-iT™ Dicer Kit

BLOCK-iT™ Kits (Invitrogen Corporation; Carlsbad, Calif.) can be usedfor fast and efficient RNAi applications. Eukaryotic cells naturallyregulate gene expression with dsRNA. A BLOCK-iT™ Dicer Kit can be usedto generate dsRNA that are then diced into siRNA, purified andtransfected into cells. The BLOCK-iT™ Dicer Kit requires no expensivesynthetic siRNAs. It also produces a pool of many siRNAs per gene, notjust one or a few, which means a higher probability of knockdown (FIGS.21, 22, and 23). A purification procedure gives a high yield of siRNAsin a transfection-ready buffer and virtually eliminates remaining longdsRNA and cleave intermediates.

BLOCK-iT™ Long RNAi Transcription Kits use a T7 TOPO linker which allowsany polymerase chain reaction (PCR) product to become a template fortranscription (FIG. 20). This mediates RNAi in invertebrates (e.g.,insects, nematodes and protozoans), some mammalian embryonic cells(undifferentiated ES cells) and many mammalian cell lines aftertreatment with Dicer/RNase III. BLOCK-iT™ Kits allows for an inexpensivealternative to siRNA oligos. Exemplary uses of BLOCK-iT™ Kits aresummarized in FIG. 24.

Kit Components and Configurations Complete Lentiviral RNAi Kit:Components of the U6 RNAi Entry Vector Kit:

Purified, BsaI-linearized pENTR/U6.2; Annealed lamin A/C control oligos:Top 5′-CACCGTGTTCTTCTGGAAGTCCAGCGAACT GGACTTCCAGAAGAACA (SEQ ID NO:9),Bottom 5′-AAAATGTTCTTCTGGA AGTCCAGTTCGCTGGACTTCCAGAAGAACAC (SEQ IDNO:10); Sequencing primers: U6 forward 5′-GGACTATCATATGCTTACCG (SEQ IDNO:11), M13 reverse 5′-CAGGAAACAGCTATGAC (SEQ ID NO:12) (Catalog No.N530-02, Invitrogen Corp., Carlsbad, Calif.); T4 DNA ligase (Catalog No.15224-025, Invitrogen Corp., Carlsbad, Calif.); 5× T4 DNA ligase buffer(Catalog No. Y90001, Invitrogen Corp., Carlsbad, Calif. Y90001); OneShotTop10 cells (Catalog No. C4040-03, Invitrogen Corp., Carlsbad, Calif.);pLenti6/RNAi/DEST; pLenti6/RNAi/U6-GW/lamAC; OneShot STBL3 cells;Virapower Bsd Lentiviral Support Kit (Catalog No. K4970-00, InvitrogenCorp., Carlsbad, Calif.); Gateway LR Clonase enzyme mix (Catalog No.11791-091, Invitrogen Corp., Carlsbad, Calif.).

Lentiviral RNAi DEST Kit:

pLenti6/RNAi/DEST; pLenti6/RNAi/U6-GW/lamAC; OneShot STBL3 cells;Gateway LR Clonase enzyme mix (Catalog No. 11791-019, Invitrogen Corp.,Carlsbad, Calif.)

REFERENCES

-   Abbas-Terki et al., Lentiviral-mediated RNA interference. Hum Gene    Ther. 13:2197-2201 (2002)-   Dirac & Bernards, Reversal of senescence in mouse fibroblasts    through lentiviral suppression of p53. J. Biol. Chem.    278:11731-11734 (2003)-   Matta et al., Use of lentiviral vectors for delivery of small    interfering RNA. Cancer Biol. Ther. 2:206-210 (2003)-   Qin et al., Inhibiting HIV-1 infection in human T cells by    lentiviral-mediated delivery of small interfering RNA against CCR5.    Proc. Natl. Acad. Sci. (USA) 100:183-188 (2003)-   Rubinson et al., A lentivirus-based system to functionally silence    genes in primary mammalian cells, stem cells and transgenic mice by    RNA interference. Nat. Genet. 33:401-406 (2003)-   Stewart et al., Lentivirus-delivered stable gene silencing by RNAi    in primary cells. RNA. 9:493-501 (2003)-   Tiscornia et al., A general method for gene knockdown in mice by    using lentiviral vectors expressing small interfering RNA. Proc.    Natl. Acad. Sci. (USA) 100:1844-1848 (2003)

TABLE 2 L × R Assay Sample Criteria Values Pass/Fail Cells only 0 cfu/μgDNA 0 cfu/μg DNA Pass No DNA 0 cfu/μg DNA 0 cfu/μg DNA Pass DEST vectoronly <1100 cfu/μg DNA 660 cfu/μg DNA Pass L × R Reaction (n = 2) ≧1.65 ×10⁶ cfu/μg DNA 2.31 × 10⁶ cfu/μg DNA Pass pUC19 only (n = 2) ≧7.5 × 10⁸cfu/μg DNA 2.53 × 10¹⁰ cfu/μg DNA Pass

TABLE 3 ccdB Assay Sample Cell Type Antibiotic Transformation EfficiencyCells Only DB3.1 Amp 0 cfu/μg DNA Kan 0 cfu/μg DNA pUC19 only (n = 4)DB3.1 Amp 7.0 × 106 cfu/μg DNA DEST vector only DB3.1 Amp 3.0 × 106cfu/μg DNA (n = 4) Cells Only TOP10 Amp 0 cfu/μg DNA Kan 0 cfu/μg DNApUC19 only (n = 4) TOP10 Amp 2.65 × 108 cfu/μg DNA DEST vector onlyTOP10 Amp 5.75 × 103 cfu/μg DNA (n = 4) Kan 0 cfu/μg DNA Fold-killing 2× 104 Pass (criteria = 1 × 104)

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising,” “consisting essentiallyof,” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions that have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed herein,optional features, modification and variation of the concepts hereindisclosed may be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein. Other aspects ofthe invention are within the following claims.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

TABLE 5 pENTRU6 Vector Nucleic Acid SequenceCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATACGCGTACCGCTAGCCAGGAAGAGTTTGTAGAAACGCAAAAAGGCCATCCGTCAGGATGGCCTTCTGCTTAGTTTGATGCCTGGCAGTTTATGGCGGGCGTCCTGCCCGCCACCCTCCGGGCCGTTGCTTCACAACGTTCAAATCCGCTCCCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTCCGACTGAGCCTTTCGTTTTATTTGATGCCTGGCAGTTCCCTACTCTCGCGTTAACGCTAGCATGGATGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTCTTAAGCTCGGGCCCCAAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAAATTGATGAGCAATGCTTTTTTATAATGCCAACTTTGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCGGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGAGACCGCGGCCGCTGGATCCGGCTTACTAAAAGCCAGATAACAGTATGCGTATTTGCGCGCTGATTTTTGCGGTATAAGAATATATACTGATATGTATACCCGAAGTATGTCAAAAAGAGGTGTGCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCGACAGCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACAACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAATCAGGAAGGGATGGCTGAGGTCGCCCGGTTTATTGAAATGAACGGCTCTTTTGCTGACGAGAACAGGGACTGGTGAAATGCAGTTTAAGGTTTACACCTATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCCGGGCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAAGGTCTCATTTTTTTTCTAGACCCAGCTTTCTTGTACAAAGTTGGCATTATAAGAAAGCATTGCTTATCAATTTGTTGCAACGAACAGGTCACTATCAGTCAAAATAAAATCATTATTTGCCATCCAGCTGATATCCCCTATAGTGAGTCGTATTACATGGTCATAGCTGTTTCCTGGCAGCTCTGGCCCGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCT TACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGAAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGAATTGGTTAATTGGTTGTAACACTGGCAGAGCATTACGCTGACTTGACGGGACGGCGCAAGCTCATGACCAAAATCCCTTAACGTGAGTTACGCGTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC ACATGTT SEQ ID NO: 1

TABLE 6 Nucleotide sequence of plasmid pLenti6/V5-DEST.AATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACATAAACGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGATAAGCTTGGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGACTCTAGAGGATCCACTAGTCCAGTGTGGTGGAATTCTGCAGATATCAACAAGTTTGTACAAAAAAGCTGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATCCAGTCACTATGGCGGCCGCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTGGATTTTGAGTTAGGATCCGGCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAAAGATCTGGATCCGGCTTACTAAAAGCCAGATAACAGTATGCGTATTTGCGCGCTGATTTTTGCGGTATAAGAATATATACTGATATGTATACCCGAAGTATGTCAAAAAGAGGTGTGCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCGACAGCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACAACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAATCAGGAAGGGATGGCTGAGGTCGCCCGGTTTATTGAAATGAACGGCTCTTTTGCTGACGAGAACAGGGACTGGTGAAATGCAGTTTAAGGTTTACACCTATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCCGGGCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAAATGTCAGGCTCCGTTATACACAGCCAGTCTGCAGGTCGACCATAGTGACTGGATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGCAAAATCTAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAGCTTTCTTGTACAAAGTGGTTGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGGCCCGCGGTTCGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTTAGTAATGAGTTTGGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGCCTTTGTCTCAAGAAGAATCCACCCTCATTGAAAGAGCAACGGCTACAATCAACAGCATCCCCATCTCTGAAGACTACAGCGTCGCCAGCGCAGCTCTCTCTAGCGACGGCCGCATCTTCACTGGTGTCAATGTATATCATTTTACTGGGGGACCTTGTGCAGAACTCGTGGTGCTGGGCACTGCTGCTGCTGCGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGAAATGAGAACAGGGGCATCTTGAGCCCCTGCGGACGGTGCCGACAGGTGCTTCTCGATCTGCATCCTGGGATCAAAGCCATAGTGAAGGACAGTGATGGACAGCCGACGGCAGTTGGGATTCGTGAATTGCTGCCCTCTGGTTATGTGTGGGAGGGCTAAGCACAATTCGAGCTCGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGAC TCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGGACGTACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGA ACAAAAGCTGGAGCTGCAAGCTTSEQ ID NO: 2

TABLE 7 Nucleotide sequence of plasmid pLenti6/V5-dTOPO ™.AATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACATAAACGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGATAAGCTTGGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGACTCTAGAGGATCCACTAGTCCAGTGTGGTGGAATTGATCCCTTCACCAAGGGCTCGAGTCTAGAGGGCCCGCGGTTCGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTTAGTAATGAGTTTGGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGCCTTTGTCTCAAGAAGAATCCACCCTCATTGAAAGAGCAACGGCTACAATCAACAGCATCCCCATCTCTGAAGACTACAGCGTCGCCAGCGCAGCTCTCTCTAGCGACGGCCGCATCTTCACTGGTGTCAATGTATATCATTTTACTGGGGGACCTTGTGCAGAACTCGTGGTGCTGGGCACTGCTGCTGCTGCGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGAAATGAGAACAGGGGCATCTTGAGCCCCTGCGGACGGTGCCGACAGGTGCTTCTCGATCTGCATCCTGGGATCAAAGCCATAGTGAAGGACAGTGATGGACAGCCGACGGCAGTTGGGATTCGTGAATTGCTGCCCTCTGGTTATGTGTGGGAGGGCTAAGCACAATTCGAGCTCGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGGACGTACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGA GCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCT GGAGCTGCAAGCTT SEQ ID NO:3

TABLE 8 Nucleotide sequence of pLenti4/V5-DEST.AATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACATAAACGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATA GAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGATAAGCTTGGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGACTCTAGAGGATCCACTAGTCCAGTGTGGTGGAATTCTGCAGATATCAACAAGTTTGTACAAAAAAGCTGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATCCAGTCACTATGGCGGCCGCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTGGATTTTGAGTTAGGATCCGGCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAAAGATCTGGATCCGGCTTACTAAAAGCCAGATAACAGTATGCGTATTTGCGCGCTGATTTTTGCGGTATAAGAAT ATATACTGATATGTATACCCGAAGTATGTCAAAAAGAGGTGTGCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCGACAGCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACAACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAATCAGGAAGGGATGGCTGAGGTCGCCCGGTTTATTGAAATGAACGGCTCTTTTGCTGACGAGAACAGGGACTGGTGAAATGCAGTTTAAGGTTTACACCTATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCCGGGCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAAATGTCAGGCTCCGTTATACACAGCCAGTCTGCAGGTCGACCATAGTGACTGGATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGCAAAATCTAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAGCTTTCTTGTACAAAGTGGTTGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGGCCCGCGGTTCGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTTAGTAATGAGTTTGGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCCCTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTGACACGTGCTACGAGATTTAAATGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGGACGTACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATG AAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTGCAAG CTT SEQ ID NO: 4

TABLE 9 Nucleotide sequence of pLenti6/UbC/V5-DEST.AATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACATAAACGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATA GAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGGATCTGGCCTCCGCGCCGGGTTTTGGCGCCTCCCGCGGGCGCCCCCCTCCTCACGGCGAGCGCTGCCACGTCAGACGAAGGGCGCAGGAGCGTCCTGATCCTTCCGCCCGGACGCTCAGGACAGCGGCCCGCTGCTCATAAGACTCGGCCTTAGAACCCCAGTATCAGCAGAAGGACATTTTAGGACGGGACTTGGGTGACTCTAGGGCACTGGTTTTCTTTCCAGAGAGCGGAACAGGCGAGGAAAAGTAGTCCCTTCTCGGCGATTCTGCGGAGGGATCTCCGTGGGGCGGTGAACGCCGATGATTATATAAGGACGCGCCGGGTGTGGCACAGCTAGTTCCGTCGCAGCCGGGATTTGGGTCGCGGTTCTTGTTTGTGGATCGCTGTGATCGTCACTTGGTGAGTAGCGGGCTGCTGGGCTGGCCGGGGCTTTCGTGGCCGCCGGGCCGCTCGGTGGGACGGAAGCGTGTGGAGAGACCGCCAAGGGCTGTAGTCTGGGTCCGCGAGCAAGGTTGCCCTGAACTGGGGGTTGGGGGGAGCGCAGCAAAATGGCGGCTGTTCCCGAGTCTTGAATGGAAGACGCTTGTGAGGCGGGCTGTGAGGTCGTTGAAACAAGGTGGGGGGCATGGTGGGCGGCAAGAACCCAAGGTCTTGAGGCCTTCGCTAATGCGGGAAAGCTCTTATTCGGGTGAGATGGGCTGGGGCACCATCTGGGGACCCTGACGTGAAGTTTGTCACTGACTGGAGAACTCGGTTTGTCGTCTGTTGCGGGGGCGGCAGTTATGCGGTGCCGTTGGGCAGTGCACCCGTACCTTTGGGAGCGCGCGCCCTCGTCGTGTCGTGACGTCACCCGTTCTGTTGGCTTATAATGCAGGGTGGGGCCACCTGCCGGTAGGTGTGCGGTAGGCTTTTCTCCGTCGCAGGACGCAGGGTTCGGGCCTAGGGTAGGCTCTCCTGAATCGACAGGCGCCGGACCTCTGGTGAGGGGAGGGATAAGTGAGGCGTCAGTTTCTTTGGTCGGTTTTATGTACCTATCTTCTTAAGTAGCTGAAGCTCCGGTTTTGAACTATGCGCTCGGGGTTGGCGAGTGTGTTTTGTGAAGTTTTTTAGGCACCTTTTGAAATGTAATCATTTGGGTCAATATGTAATTTTCAGTGTTAGACTAGTAAATTGTCCGCTAAATTCTGGCCGTTTTTGGCTTTTTTGTTAGACGAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCTGCAGATATCAACAAGTTTGTACAAAAAAGCTGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATCCAGTCACTATGGCGGCCGCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTGGATTTTGAGTTAGGATCCGGCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCC GCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAAAGATCTGGATCCGGCTTACTAAAAGCCAGATAACAGTATGCGTATTTGCGCGCTGATTTTTGCGGTATAAGAATATATACTGATATGTATACCCGAAGTATGTCAAAAAGAGGTGTGCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCGACAGCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACAACCATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAATCAGGAAGGGATGGCTGAGGTCGCCCGGTTTATTGAAATGAACGGCTCTTTTGCTGACGAGAACAGGGACTGGTGAAATGCAGTTTAAGGTTTACACCTATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCCGGGCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAAATGTCAGGCTCCGTTATACACAGCCAGTCTGCAGGTCGACCATAGTGACTGGATATGTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGCAAAATCTAATTTAATATATTGATATTTATATCATTTTACGTTTCTCGTTCAGCTTTCTTGTACAAAGTGGTTGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGGCCCGCGGTTCGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCGTACCGGTTAGTAATGAGTTTGGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTA TGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGCCTTTGTCTCAAGAAGAATCCACCCTCATTGAAAGAGCAACGGCTACAATCAACAGCATCCCCATCTCTGAAGACTACAGCGTCGCCAGCGCAGCTCTCTCTAGCGACGGCCGCATCTTCACTGGTGTCAATGTATATCATTTTACTGGGGGACCTTGTGCAGAACTCGTGGTGCTGGGCACTGCTGCTGCTGCGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGAAATGAGAACAGGGGCATCTTGAGCCCCTGCGGACGGTGCCGACAGGTGCTTCTCGATCTGCATCCTGGGATCAAAGCCATAGTGAAGGACAGTGATGGACAGCCGACGGCAGTTGGGATTCGTGAATTGCTGCCCTCTGGTTATGTGTGGGAGGGCTAAGCACAATTCGAGCTCGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGGACGTACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGG GGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGA ACAAAAGCTGGAGCTGCAAGCTTSEQ ID NO: 5

TABLE 10 Nucleotide sequence of plasmid pLP1.TTGGCCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCCCTCGAAGCTTACATGTGGTACCGAGCTCGGATCCTGAGAACTTCAGGGTGAGTCTATGGGACCCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTTCATGTCATAGGAAGGGGAGAAGTAACAGGGTACACATATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGCACGTGAGATCTGAATTCGAGATCTGCCGCCGCCATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGAAAAAAGCACAGCAAGCAGCAGCTGACACAGGACACAGCAATCAGGTCAGCCAAAATTACCCTATAGTGCAGAACATCCAGGGGCAAATGGTACATCAGGCCATATC ACCTAGAACTTTAAATGCATGGGTAAAAGTAGTAGAAGAGAAGGCTTTCAGCCCAGAAGTGATACCCATGTTTTCAGCATTATCAGAAGGAGCCACCCCACAAGATTTAAACACCATGCTAAACACAGTGGGGGGACATCAAGCAGCCATGCAAATGTTAAAAGAGACCATCAATGAGGAAGCTGCAGAATGGGATAGAGTGCATCCAGTGCATGCAGGGCCTATTGCACCAGGCCAGATGAGAGAACCAAGGGGAAGTGACATAGCAGGAACTACTAGTACCCTTCAGGAACAAATAGGATGGATGACACATAATCCACCTATCCCAGTAGGAGAAATCTATAAAAGATGGATAATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTACCAGCATTCTGGACATAAGACAAGGACCAAAGGAACCCTTTAGAGACTATGTAGACCGATTCTATAAAACTCTAAGAGCCGAGCAAGCTTCACAAGAGGTAAAAAATTGGATGACAGAAACCTTGTTGGTCCAAAATGCGAACCCAGATTGTAAGACTATTTTAAAAGCATTGGGACCAGGAGCGACACTAGAAGAAATGATGACAGCATGTCAGGGAGTGGGGGGACCCGGCCATAAAGCAAGAGTTTTGGCTGAAGCAATGAGCCAAGTAACAAATCCAGCTACCATAATGATACAGAAAGGCAATTTTAGGAACCAAAGAAAGACTGTTAAGTGTTTCAATTGTGGCAAAGAAGGGCACATAGCCAAAAATTGCAGGGCCCCTAGGAAAAAGGGCTGTTGGAAATGTGGAAAGGAAGGACACCAAATGAAAGATTGTACTGAGAGACAGGCTAATTTTTTAGGGAAGATCTGGCCTTCCCACAAGGGAAGGCCAGGGAATTTTCTTCAGAGCAGACCAGAGCCAACAGCCCCACCAGAAGAGAGCTTCAGGTTTGGGGAAGAGACAACAACTCCCTCTCAGAAGCAGGAGCCGATAGACAAGGAACTGTATCCTTTAGCTTCCCTCAGATCACTCTTTGGCAGCGACCCCTCGTCACAATAAAGATAGGGGGGCAATTAAAGGAAGCTCTATTAGATACAGGAGCAGATGATACAGTATTAGAAGAAATGAATTTGCCAGGAAGATGGAAACCAAAAATGATAGGGGGAATTGGAGGTTTTATCAAAGTAAGACAGTATGATCAGATACTCATAGAAATCTGCGGACATAAAGCTATAGGTACAGTATTAGTAGGACCTACACCTGTCAACATAATTGGAAGAAATCTGTTGACTCAGATTGGCTGCACTTTAAATTTTCCCATTAGTCCTATTGAGACTGTACCAGTAAAATTAAAGCCAGGAATGGATGGCCCAAAAGTTAAACAATGGCCATTGACAGAAGAAAAAATAAAAGCATTAGTAGAAATTTGTACAGAAATGGAAAAGGAAGGAAAAATTTCAAAAATTGGGCCTGAAAATCCATACAATACTCCAGTATTTGCCATAAAGAAAAAAGACAGTACTAAATGGAGAAAATTAGTAGATTTCAGAGAACTTAATAAGAGAACTCAAGATTTCTGGGAAGTTCAATTAGGAATACCACATCCTGCAGGGTTAAAACAGAAAAAATCAGTAACAGTACTGGATGTGGGCGATGCATATTTTTCAGTTCCCTTAGATAAAGACTTCAGGAAGTATACTGCATTTACCATACCTAGTATAAACAATGAGACACCAGGGATTAGATAT CAGTACAATGTGCTTCCACAGGGATGGAAAGGATCACCAGCAATATTCCAGTGTAGCATGACAAAAATCTTAGAGCCTTTTAGAAAACAAAATCCAGACATAGTCATCTATCAATACATGGATGATTTGTATGTAGGATCTGACTTAGAAATAGGGCAGCATAGAACAAAAATAGAGGAACTGAGACAACATCTGTTGAGGTGGGGATTTACCACACCAGACAAAAAACATCAGAAAGAACCTCCATTCCTTTGGATGGGTTATGAACTCCATCCTGATAAATGGACAGTACAGCCTATAGTGCTGCCAGAAAAGGACAGCTGGACTGTCAATGACATACAGAAATTAGTGGGAAAATTGAATTGGGCAAGTCAGATTTATGCAGGGATTAAAGTAAGGCAATTATGTAAACTTCTTAGGGGAACCAAAGCACTAACAGAAGTAGTACCACTAACAGAAGAAGCAGAGCTAGAACTGGCAGAAAACAGGGAGATTCTAAAAGAACCGGTACATGGAGTGTATTATGACCCATCAAAAGACTTAATAGCAGAAATACAGAAGCAGGGGCAAGGCCAATGGACATATCAAATTTATCAAGAGCCATTTAAAAATCTGAAAACAGGAAAGTATGCAAGAATGAAGGGTGCCCACACTAATGATGTGAAACAATTAACAGAGGCAGTACAAAAAATAGCCACAGAAAGCATAGTAATATGGGGAAAGACTCCTAAATTTAAATTACCCATACAAAAGGAAACATGGGAAGCATGGTGGACAGAGTATTGGCAAGCCACCTGGATTCCTGAGTGGGAGTTTGTCAATACCCCTCCCTTAGTGAAGTTATGGTACCAGTTAGAGAAAGAACCCATAATAGGAGCAGAAACTTTCTATGTAGATGGGGCAGCCAATAGGGAAACTAAATTAGGAAAAGCAGGATATGTAACTGACAGAGGAAGACAAAAAGTTGTCCCCCTAACGGACACAACAAATCAGAAGACTGAGTTACAAGCAATTCATCTAGCTTTGCAGGATTCGGGATTAGAAGTAAACATAGTGACAGACTCACAATATGCATTGGGAATCATTCAAGCACAACCAGATAAGAGTGAATCAGAGTTAGTCAGTCAAATAATAGAGCAGTTAATAAAAAAGGAAAAAGTCTACCTGGCATGGGTACCAGCACACAAAGGAATTGGAGGAAATGAACAAGTAGATAAATTGGTCAGTGCTGGAATCAGGAAAGTACTATTTTTAGATGGAATAGATAAGGCCCAAGAAGAACATGAGAAATATCACAGTAATTGGAGAGCAATGGCTAGTGATTTTAACCTACCACCTGTAGTAGCAAAAGAAATAGTAGCCAGCTGTGATAAATGTCAGCTAAAAGGGGAAGCCATGCATGGACAAGTAGACTGTAGCCCAGGAATATGGCAGCTAGATTGTACACATTTAGAAGGAAAAGTTATCTTGGTAGCAGTTCATGTAGCCAGTGGATATATAGAAGCAGAAGTAATTCCAGCAGAGACAGGGCAAGAAACAGCATACTTCCTCTTAAAATTAGCAGGAAGATGGCCAGTAAAAACAGTACATACAGACAATGGCAGCAATTTCACCAGTACTACAGTTAAGGCCGCCTGTTGGTGGGCGGGGATCAAGCAGGAATTTGGCATTCCCTACAATCCCCAAAGTCAAGGAGTAATAGAATCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTGGAAAGGACCAGCAAAGCTCCTCTGGAAAGGTGAAGGGGCAGTAGTAATACAAGATAATAGTGACATAAAAGTAGTGCCAAGAAGAAAAGCAAAGATCATCAGGGATTATGGAAAACAGATGGCAGGTGATGATTGTGTGGCAAGTAGACAGGATGAGGATTAACACATGGAATTCCGGAGCGGCCGCAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTCCGCGGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAATGCCCTGGCCCACAAGTATCACTAAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAATGATGTATTTAAATTATTTCTGAATATTTTACTAAAAAGGGAATGTGGGAGGTCAGTGCATTTAAAACATAAAGAAATGAAGAGCTAGTTCAAACCTTGGGAAAATACACTATATCTTAAACTCCATGAAAGAAGGTGAGGCTGCAAACAGCTAATGCACATTGGCAACAGCCCCTGATGCCTATGCCTTATTCATCCCTCAGAAAAGGATTCAAGTAGAGGCTTGATTTGGAGGTTAAAGTTTTGCTATGCTGTATTTTACATTACTTATTGTTTTAGCTGTCCTCATGAATGTCTTTTCACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTGACTCCACTCAGTTCTCTTGCTTAGAGATACCACCTTTCCCCTGAAGTGTTCCTTCCATGTTTTACGGCGAGATGGTTTCTCCTCGCCTGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTTATAGAGGTCTACTTGAAGAAGGAAAAACAGGGGGCATGGTTTGACTGTCCTGTGAGCCCTTCTTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCCCTCGACATGGCAGTCTAGCACTAGTGCGGCCGCAGATCTGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGGGATCCCCTGAGGGGGCCCCCATGGGCTAGAGGATCCGGCCTCGGCCTCTGCATAAATA AAAAAAATTAGTCAGCCATGAGCSEQ ID NO: 6

TABLE 11 Nucleotide sequence of plasmid pLP2.AATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACAATAAACGCCATTTGACCATTCACCACATTGGTGTGCACCTCCAAGCTCGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCCCTCGAAGCTAGTCGATTAGGCATCTCCTATGGCAGGAAGAAGCGGAGACAGCGACGAAGACCTCCTCAAGGCAGTCAGACTCATCAAGTTTCTCTATCAAAGCAACCCACCTCCCAATCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCCTTAGCACTTATCTGGGACGATCTGCGGAGCCTGTGCCTCTTCAGCTACCACCGCTTGAGAGACTTACTCTTGATTGTAACGAGGATTGTGGAACTTCTGGGACGCAGGGGGTGGGAAGCCCTCAAATATTGGTGGAATCTCCTACAATATTGGAGTCAGGAGCTAAAGAATAGTGCTGTTAGCTTGCTCAATGCCACAGCTATAGCAGTAGCTGAGGGGACAGATAGGGTTATAGAAGTAGTACAAGAAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTGATCTGAGCCTGGGAGATCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCAGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACATGATTACGAATTCGATGTACGGGCCAGATATACGCGTATCTGAGGGGACTAGGGTGTGTTTAGGCGAAAAGCGGGGCTTCGGTTGTACGCGGTTAGGAGTCCCCTCAGGATATAGTAGTTTCGCTTTTGCATAGGGAGGGGGA SEQ ID NO: 7

TABLE 12 Nucleotide sequence of plasmid pLP/VSVG.TTGGCCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCCCTCGAAGCTTACATGTGGTACCGAGCTCGGATCCTGAGAACTTCAGGGTGAGTCTATGGGACCCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTTCATGTCATAGGAAGGGGAGAAGTAACAGGGTACACATATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGCACGTGAGATCTGAATTCTGACACTATGAAGTGCCTTTTGTACTTAGCCTTTTTATTCATTGGGGTGAATTGCAAGTTCACCATAGTTTTTCCACACAACCAAAAAGGAAACTGGAAAAATGTTCCTTCTAATTACCATTATTGCCCGTCAAGCTCAGATTTAAATTGGCATAATGACTTAATAGGCACAGCCTTACAAGTCAAAATGCCCAAGAGTCACAAGGCTATTCAAGCAGACGGTTGGATGTGTCATGCTTCCAAATGGGTCACTACTTGTGATTTCCGCTGGTATGGACCGAAGTATATAACACATTCCATCCGATCCTTCACTCCATCTGTAGAACAATGCAAGGAAAGCATTGAACAAACGAAACAAGGAACTTGGCTGAATCCAGGCTTCCCTCCTCAAAGTTGTGGATATGCAACTGTGACGGATGCCGAAGCAGTGATTGTCCAGGTGACTCCTCACCATGTGCTGGTTGATGAATACACAGGAGAATGGGTTGATTCACAGTTCATCAACGGAAAATGCAGCAATTACATATGCCCCACTGTCCATAACTCTACAACCTGGCATTCTGACTATAAGGTCAAAGGGCTATGTGATTCTAACCTCATTTCCATGGACATCACCTTCTTCTCAGAGGACGGAGAGCTATCATCCCTGGGAAAGGAGGGCACAGGGTTCAGAAGTAACTACTTTGCTTATGAAACTGGAGGCAAGGCCTGCAAAATGCAATACTGCAAGCATTGGGGAGTCAGACTCCCATCAGGTGTCTGGTTCGAGATGGCTGATAAGGATCTCTTTGCTGCAGCCAGATTCCCTGAATGCCCAGAAGGGTCAAGTATCTCTGCTCCATCTCAGACCTCAGTGGATGTAAGTCTAATTCAGGACGTTGAGAGGATCTTGGATTATTCCCTCTGCCAAGAAACCTGGAGCAAAATCAGAGCGGGTCTTCCAATCTCTCCAGTGGATCTCAGCTATCTTGCTCCTAAAAACCCAGGAACCGGTCCTGCTTTCACCATAATCAATGGTACCCTAAAATACTTTGAGACCAGATACATCAGAGTCGATATTGCTGCTCCAATCCTCTCAAGAATGGTCGGAATGATCAGTGGAACTACCACAGAAAGGGAACTGTGGGATGACTGGGCACCATATGAAGACGTGGAAATTGGACCCAATGGAGTTCTGAGGACCAGTTCAGGATATAAGTTTCCTTTATACATGATTGGACATGGTATGTTGGACTCCGATCTTCATCTTAGCTCAAAGGCTCAGGTGTTCGAACATCCTCACATTCAAGACGCTGCTTCGCAACTTCCTGATGATGAGAGTTTATTTTTTGGTGATACTGGGCTATCCAAAAATCCAATCGAGCTTGTAGAAGGTTGGTTCAGTAGTTGGAAAAGCTCTATTGCCTCTTTTTTCTTTATCATAGGGTTAATCATTGGACTATTCTTGGTTCTCCGAGTTGGTATCCATCTTTGCATTAAATTAAAGCACACCAAGAAAAGACAGATTTATACAGACATAGAGATGAACCGACTTGGAAAGTAACTCAAATCCTGCACAACAGATTCTTCATGTTTGGACCAAATCAACTTGTGATACCATGCTCAAAGAGGCCTCAATTATATTTGAGTTTTTAATTTTTATGAAAAAAAAAAAAAAAAACGGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAATGCCCTGGCCCACAAGTATCACTAAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAATGATGTATTTAAATTATTTCTGAATATTTTACTAAAAAGGGAATGTGGGAGGTCAGTGCATTTAAAACATAAAGAAATGAAGAGCTAGTTCAAACCTTGGGAAAATACACTATATCTTAAACTCCATGAAAGAAGGTGAGGCTGCAAACAGCTAATGCACATTGGCAACAGCCCCTGATGCCTATGCCTTATTCATCCCTCAGAAAAGGATTCAAGTAGAGGCTTGATTTGGAGGTTAAAGTTTTGCTATGCTGTATTTTACATTACTTATTGTTTTAGCTGTCCTCATGAATGTCTTTTCACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTGACTCCACTCAGTTCTCTTGCTTAGAGATACCACCTTTCCCCTGAAGTGTTCCTTCCATGTTTTACGGCGAGATGGTTTCTCCTCGCCTGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTTATAGAGGTCTACTTGAAGAAGGAAAAACAGGGGGCATGGTTTGACTGTCCTGTGAGCCCTTCTTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCCCTCGACATGGCAGTCTAGCACTAGTGCGGCCGCAGATCTGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGGGATCCCCTGAGGGGGCCCCCATGGGCTAGAGGATCCGGCCTCGGCCTCTGCATAAATAAAAAAAATTAGTCAGCCATGAGC SEQ ID NO: 8

1. A method of producing an RNA molecule for use as an interfering RNAcomprising: (a) identifying one or more target nucleic acid sequences;(b) preparing one or more nucleic acid molecules which encode one ormore interfering RNAs, wherein said interfering RNAs bind to said one ormore target nucleic acid sequences; (c) combining (i) one or more firstnucleic acid molecules encoding one or more interfering RNAs flanked byone or more first type IIs restriction enzyme recognition sites; (ii)one or more second nucleic acid molecules comprising one or moreselectable markers flanked by one or more second type IIs restrictionenzyme recognition sites; and (iii) one or more site-specific type IIsrestriction enzymes; and (d) incubating said combination underconditions sufficient to join one or more of said nucleic acid moleculesencoding one or more interfering RNAs and one or more of said secondnucleic acid molecules, thereby producing one or more desired productnucleic acid molecules; (e) inserting said one or more product nucleicacid molecules into a host cell; and (f) expressing said one or moreinterfering RNAs in said host cell. 2-5. (canceled)
 6. The method ofclaim 1, wherein said one or more selectable markers comprises at leastone DNA segment encoding an element selected from the group consistingof an antibiotic resistance gene, a gene that encodes a fluorescentprotein, an auxotrophic marker, a toxic gene and a phenotypic marker. 7.The method of claim 6, wherein said antibiotic resistance gene isselected from the group consisting of a chloramphenicol resistance gene,an ampicillin resistance gene, a tetracycline resistance gene, a Zeocinresistance gene, a spectinomycin resistance gene and a kanamycinresistance gene.
 8. The method of claim 6, wherein said toxic gene isselected from the group consisting of a ccdB gene, a gene encoding a tusprotein, a kicB gene, a sacB gene, an ASK1 gene, a ΦX174 E gene and aDpnI gene.
 9. The method of claim 1, wherein said first nucleic acidmolecule and/or said second nucleic acid molecule further comprises oneor more recombination sites.
 10. The method of claim 9, wherein saidfirst nucleic acid molecule and/or said second nucleic acid moleculefurther comprises one or more topoisomerase recognition sites and/or oneor more topoisomerases.
 11. The method of claim 10, wherein said firstnucleic acid molecule and/or said second nucleic acid molecule comprisestwo or more recombination sites.
 12. The method of claim 11, whereinsaid topoisomerase recognition site, if present, is flanked by said twoor more recombination sites.
 13. The method of claim 12, wherein saidrecombination sites are selected from the group consisting of attBsites, attP sites, attL sites, attR sites, lox sites, psi sites, tnpIsites, dif sites, cer sites, frt sites, and mutants, variants andderivatives thereof.
 14. The method of claim 10, wherein saidtopoisomerase recognition site, if present, is recognized and bound by atype I topoisomerase.
 15. The method of claim 14, wherein said type Itopoisomerase is a type IB topoisomerase.
 16. The method of claim 15,wherein said type IB topoisomerase is selected from the group consistingof eukaryotic nuclear type I topoisomerase and a poxvirus topoisomerase.17. The method of claim 1, wherein said expressed interfering RNA isbetween 35-60 nucleotides in length.
 18. The method of claim 17, whereinsaid expressed interfering RNA forms a hairpin loop. 19-20. (canceled)21. A vector comprising: (a) one or more toxic genes; (b) one or moretype IIs restriction enzyme recognition sites; and (c) one or moresite-specific recombination sites.
 22. The vector of claim 21, whereinsaid type IIs restriction enzyme recognition sites are selected from thegroup consisting of BsaI, BbsI, BbvII, BsmAI, BspMI, Eco3II, BsmBI,BaeI, FokI, HgaI, SlaNI and Sth132I.
 23. The vector of claim 21, whereinsaid recombination sites are selected from the group consisting of attBsites, attP sites, attL sites, attR sites, lox sites, psi sites, tnpIsites, dif sites, cer sites, frt sites, and mutants, variants andderivatives thereof.
 24. The vector of claim 21, wherein said vectorfurther comprises one or more topoisomerase recognition sites and/or oneor more topoisomerases.
 25. The vector of claim 24, wherein saidmolecule comprises two or more recombination sites.
 26. The vector ofclaim 24, wherein said topoisomerase recognition site, if present, isflanked by said two or more recombination sites.
 27. The vector of claim24, wherein said topoisomerase recognition site, if present, isrecognized and bound by a type I topoisomerase.
 28. The vector of claim27, wherein said type I topoisomerase is a type IB topoisomerase.
 29. Amethod of regulating the expression of one or more genes in a transgeniccell or a transgenic animal using interfering RNA, comprising: (a)identifying one or more target nucleic acid sequences in said cell oranimal; (b) preparing one or more nucleic acid molecules which encodeone or more interfering RNAs, wherein said interfering RNAs bind to saidone or more target nucleic acid sequences; (c) combining (i) one or morefirst nucleic acid molecules encoding one or more interfering RNAsflanked by one or more first type IIs restriction enzyme recognitionsites; (ii) one or more second nucleic acid molecules comprising one ormore selectable markers flanked by one or more second type IIsrestriction enzyme recognition sites; and (iii) one or moresite-specific type IIs restriction enzymes; and (d) incubating saidcombination under conditions sufficient to join one or more of said oneor more nucleic acid molecules encoding one or more interfering RNAs andone or more of said second nucleic acid molecules, thereby producing oneor more desired product nucleic acid molecules; (e) inserting said oneor more interfering RNA-containing product nucleic acid molecules intosaid cell or one or more cells of said animal, under conditions suchthat said one or more interfering RNAs bind to said one or more targetnucleic acid sequences, thereby regulating expression of said one ormore genes.
 30. The method of claim 29, wherein said expressedinterfering RNA is between 35-60 nucleotides in length.
 31. The methodof claim 30, wherein said expressed interfering RNA forms a hairpinloop. 32-33. (canceled)
 34. The method of claim 29, wherein saidregulation results in decreased expression of said one or more genes.